Methods for immunizing pre-immune subjects against respiratory syncytial virus (rsv)

ABSTRACT

The invention provides methods for using virus-like particle (VLP) vaccines containing a stabilized pre-fusion respiratory syncytial virus (RSV) F protein to stimulate RSV neutralizing antibodies in pre-immune subjects. In one embodiment, the invention provides a method for immunizing a mammalian subject in need of immunizing against Respiratory Syncytial virus (RSV) infection, comprising, a) providing i) a pre-immune mammalian subject containing RSV neutralizing antibodies, ii) a first composition comprising recombinant chimeric Newcastle disease virus-like particles (ND VLPs), that contain a chimeric protein comprising, in operable combination, 1) stabilized pre-fusion RSV F protein ectodomain, 2) transmembrane (TM) domain of NDV F protein, and 3) cytoplasmic (CT) domain of NDV F protein, and b) administering an immunologically effective amount of the first composition to the pre-immune subject to produce an immunized subject that comprises an increase in the level of the RSV neutralizing antibodies compared to the level of RSV neutralizing antibodies in the pre-immune subject. In one embodiment, the level of the RSV neutralizing antibodies in the pre-immune subject does not prevent RSV infection of the pre-immune subject.

This application is a continuation application of, and claims priorityto, copending U.S. application Ser. No. 16/339,219 filed Apr. 3, 2019,which is the U.S. National stage filing under 35 U.S.C § 371 of, andwhich claims priority to, co-pending PCT Application No. PCT/US17/52247,filed Sep. 19, 2017, which claims priority under 35 U.S.C. § 119(e) toU.S. provisional Application Ser. No. 62/403,229, filed Oct. 3, 2016,now abandoned, the entire contents of each of which is hereinincorporated by reference.

A Sequence Listing has been submitted in an ASCII text file named“18737” created on Dec. 9, 2020, consisting of 38,132 bytes, the entirecontent of which is herein incorporated by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numberAI114809 awarded by the National Institutes of Health (NIH). Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention provides methods for using virus-like particle (VLP)vaccines containing a stabilized pre-fusion respiratory syncytial virus(RSV) F protein to stimulate RSV neutralizing antibodies in pre-immunesubjects. In one embodiment, the invention provides a method forimmunizing a mammalian subject in need of immunizing against RespiratorySyncytial virus (RSV) infection, comprising, a) providing i) apre-immune mammalian subject containing RSV neutralizing antibodies, ii)a first composition comprising recombinant chimeric Newcastle diseasevirus-like particles (ND VLPs), that contain a chimeric proteincomprising, in operable combination, 1) stabilized pre-fusion RSV Fprotein ectodomain, 2) transmembrane (TM) domain of NDV F protein, and3) cytoplasmic (CT) domain of NDV F protein, and b) administering animmunologically effective amount of the first composition to thepre-immune subject to produce an immunized subject that comprises anincrease in the level of the RSV neutralizing antibodies compared to thelevel of RSV neutralizing antibodies in the pre-immune subject. In oneembodiment, the level of the RSV neutralizing antibodies in thepre-immune subject does not prevent RSV infection of the pre-immunesubject.

BACKGROUND OF THE INVENTION

Respiratory syncytial virus (RSV) is the single most important cause ofacute viral respiratory disease in infants and young children (1)frequently resulting in hospitalization and in significant mortalityrates particularly in developing countries. RSV infection alsosubstantially impacts elderly and immunocompromised populations (2-5).In addition, RSV infections result in considerable morbidity in normaladult populations (6). Despite the significance of RSV disease in manydifferent populations, there are no vaccines available.

Numerous vaccine candidates have been characterized in preclinical andclinical studies spanning five decades but none have been licensed. Fourinterrelated problems have uniquely hindered

RSV vaccine development. First is safety. Using classical methods forinactivated vaccine preparation, a formalin-inactivated preparation ofpurified virus (FI-RSV) not only failed to protect infants frominfection, but also unexpectedly resulted in enhanced respiratorydisease (ERD) upon subsequent infection with RSV (reviewed in (7-10)).The mechanisms responsible for this unusual response to a classicallyprepared vaccine are not completely understood even after decades ofresearch using animal models.

A second major problem has been a lack of understanding of requirementsfor generation of high titers of neutralizing antibodies. A likelyreason for the failure of most vaccine candidates is that they did notcontain the appropriate form of the F protein. Like other paramyxovirusF proteins, the RSV F protein is folded into a metastable pre-fusionconformation and upon fusion activation refolds into the post-fusionconformation, which is structurally very different from the pre-fusionform (11-18). The pre-fusion form of F protein may be the most effectivein stimulating optimally neutralizing antibodies. Indeed, the pre-fusionform contains unique epitopes, such as site φ, missing from the postfusion form. Antibodies to site φ neutralize virus at far lowerconcentrations than antibodies specific to sites common to both the pre-and post-F forms, sites I, II, and IV (18, 19). What was not recognizeduntil recently is that the pre-fusion form of the RSV F protein isunusually unstable and that most vaccine candidates contained primarilythe post fusion form. In spite of this, it has been argued, by some,that a post-fusion F protein will stimulate protection (20) and thisform of F protein is now in clinical trials. In contrast, Magro, et alreported results, confirmed by Ngwuta, et al (21), that mostneutralizing antibodies in human or rabbit anti-RSV immune sera do notbind to the post-fusion F protein but do bind to the pre-fusion Fprotein (22) suggesting that the majority of effective neutralizingantibody binding sites reside on the pre-fusion F protein. Furthermore,McLellan, et al (19) have shown that a soluble form of pre-fusion of Fprotein, stabilized by mutation (DS-Cav mutant F protein), stimulatedsignificantly higher neutralizing antibody titers, in both mice andnon-human primates, than those stimulated by post fusion forms.

A third problem is that the vast majority of the population hasexperienced RSV infection by 2 years of age (23). While RSV infectiondoes not stimulate effective long-term protective immunity, anypreexisting immunity could potentially impact the effectiveness of avaccine. Thus any vaccine candidate must stimulate high titers ofneutralizing antibody in the face of this preexisting immunity, a topicthat has not been widely addressed

A fourth related problem is a lack of understanding of requirements forthe induction of effective long-lived and memory responses to RSV. Oneof the hallmarks of RSV infection is the observation that humansexperience repeated infection caused by the same virus sero-groupmultiple times over several years or even within the same season (24,25). Indeed, Pulendran and Ahmed (26) have noted that a successful RSVvaccine, in contrast to most vaccines, must stimulate better immuneresponses than natural infection. To date, the analysis of induction oflong-term protective responses to vaccine candidates has not been theprimary focus of RSV vaccine development.

Despite the significance of RSV disease in many different populations,the above problems have resulted in the lack of availability of RSVvaccine. What are needed are compositions methods for generating RSVneutralizing antibodies to vaccinate subjects with preexisting immunity.

SUMMARY OF THE INVENTION

In one embodiment, the invention provides a method for vaccinatingand/or immunizing a mammalian subject in need thereof, comprising, a)providing i) a first pre-immune mammalian subject containing RespiratorySyncytial Virus (RSV) neutralizing antibodies, ii) a first compositioncomprising a recombinant chimeric Newcastle Disease virus-like particle(ND VLP) that contains a chimeric protein comprising, in operablecombination, 1) stabilized pre-fusion RSV F protein ectodomain, 2)transmembrane (TM) domain of NDV F protein, and 3) cytoplasmic (CT)domain of NDV F protein, and b) administering an immunologicallyeffective amount of the first composition to the first pre-immunesubject under conditions for producing an immunized subject comprisingan increase in the level of the RSV neutralizing antibodies compared tothe level of RSV neutralizing antibodies in the first pre-immunesubject.

In a further embodiment, the invention provides a method for immunizinga mammalian subject, comprising, a) providing i) a first pre-immunemammalian subject containing RSV neutralizing antibodies, ii) a firstcomposition comprising recombinant chimeric Newcastle Disease virus-likeparticles (ND VLPs) that contain a chimeric protein comprising, inoperable combination, 1) stabilized pre-fusion RSV F protein ectodomain,2) transmembrane (TM) domain of NDV F protein, and 3) cytoplasmic (CT)domain of NDV F protein, and b) administering an immunologicallyeffective amount of the first composition to the first pre-immunemammalian subject thereby immunizing said subject under conditions wherethe level of the RSV neutralizing antibodies in said immunized mammaliansubject is increased. In one preferred embodiment, the level of the RSVneutralizing antibodies in the first pre-immune subject does not preventRSV infection of the first pre-immune subject.

In another embodiment, the invention provides a method for immunizing amammalian subject, comprising, a) providing i) a first pre-immunemammalian subject containing RSV neutralizing antibodies, ii) a firstcomposition comprising recombinant chimeric Newcastle Disease virus-likeparticles (ND VLPs) that contain a chimeric protein comprising, inoperable combination, 1) stabilized pre-fusion RSV F protein ectodomain,2) transmembrane (TM) domain of NDV F protein, and 3) cytoplasmic (CT)domain of NDV F protein, and b) administering an immunologicallyeffective amount of the first composition to the first pre-immunemammalian subject (e.g., to produce a first immunized mammaliansubject), wherein said administering is under conditions that increasethe level of the RSV neutralizing antibodies in said first immunizedmammalian subject. In one preferred embodiment, the level of the RSVneutralizing antibodies in the first pre-immune subject does not preventRSV infection of the first pre-immune subject. Thus, in someembodiments, the method comprises determining the presence of RSVinfection in one or both of the first pre-immune subject and the firstimmunized subject.

In a particular embodiment, the level of the RSV neutralizing antibodiesin the first immunized subject reduces RSV infection of the firstimmunized subject compared to the first pre-immune subject. Thus, insome embodiments, the method comprises detecting the level of RSVinfection. In a further embodiment, the level of the RSV neutralizingantibodies in the first immunized subject reduces one or more symptomsof RSV infection. Thus, in some embodiments, the method comprisesdetermining the level of RSV neutralizing antibodies in one or both ofthe first immunized subject and second immunized subject, and/ordetermining the presence and/or absence of one or more symptoms of RSVinfection in one or both of the first immunized subject and the secondimmunized subject. In yet another embodiment, the level of the RSVneutralizing antibodies in the first immunized subject reducessusceptibility of the first immunized subject to RSV infection comparedto the first pre-immune subject. Thus, in some embodiments, the methodcomprises determining susceptibility of the first immunized subject toRSV infection. In a further embodiment, the level of the RSVneutralizing antibodies in the first immunized subject reducestransmission of RSV infection from the first immunized subject. Thus, insome embodiments, the method comprises determining transmission of RSVinfection from the first immunized subject. In one embodiment, theincrease in the level of the RSV neutralizing antibodies in the firstimmunized subject is at least 100% compared to the level of RSVneutralizing antibodies in the first pre-immune subject. In a particularembodiment, the first immunized subject comprises an increase in thelevel of the RSV neutralizing antibodies compared to the level of RSVneutralizing antibodies in second immunized subject, wherein said secondimmunized subject is a second pre-immune subject that is infected withRSV. In one embodiment, the increase in the level of the RSVneutralizing antibodies in the first immunized subject is at least 100%compared to the level of RSV neutralizing antibodies in the secondpre-immune subject that is infected with RSV. In another embodiment, thefirst immunized subject comprises an increase in the level of the RSVneutralizing antibodies compared to the level of RSV neutralizingantibodies in a second pre-immune subject that is immunized with asecond composition comprising chimeric ND VLPs that contain, in operablecombination 1) stabilized post-fusion RSV F protein ectodomain, 2)transmembrane domain of NDV F protein, and 3) cytoplasmic domain of NDVF protein. In a particular embodiment, the increase in the level of theRSV neutralizing antibodies in the first immunized subject is at least100% compared to the second pre-immune subject that is treated with thesecond composition. In a further embodiment, the chimeric ND VLPsfurther comprise, in operable combination, foldon sequence listed as SEQID NO:14. In another embodiment, the level of the RSV neutralizingantibodies after a single administration of a dose of the firstcomposition to the first pre-immune subject is substantially the same asthe level of RSV neutralizing antibodies after twice administering thedose of the first composition to a control naïve subject. In anotherembodiment, the administering step is carried out at least once. In yeta further embodiment, the method further comprises comparing the levelof the RSV neutralizing antibodies in the first immunized subject to thelevel of the RSV neutralizing antibodies in one or more test subjectsselected from the group consisting of a) the first pre-immune subject,b) a second pre-immune subject that is infected with RSV, c) a secondpre-immune subject that is treated with a second composition comprisingchimeric ND VLPs that contain, in operable combination 1) stabilizedpost-fusion RSV F protein ectodomain, 2) transmembrane domain of NDV Fprotein, and 3) cytoplasmic domain of NDV F protein, wherein detectingan increase in the level of the RSV neutralizing antibodies in the firstimmunized subject compared to the level of the RSV neutralizingantibodies in the one or more test subjects indicates that the firstimmunized subject is immunized against the RSV infection. In aparticular embodiment, the method further comprises detecting in thefirst immunized subject a reduction in one or more of (a) level of RSVinfection, (b) one or more symptoms of RSV infection, (c) susceptibilityto RSV infection, and (d) transmission of RSV infection, compared to thefirst pre-immune subject.

In one embodiment, the level, in the first immunized subject, ofimmunoglobulin G (IgG) that is specific for the stabilized pre-fusionRSV F protein ectodomain F is lower than the level, in one or both ofthe first immunized subject and the second immunized subject, ofimmunoglobulin G (IgG) that is specific for the stabilized post-fusionRSV F protein ectodomain F. In a particular embodiment, the ND VLPfurther comprises, in operable combination, CT domain of NDV HN protein,TM domain of NDV HN protein, and RSV G ectodomain protein. In anotherembodiment, the level of antibody that is specific for the RSV Gectodomain protein in the first immunized subject after a singleadministration of the first composition is higher than the level ofantibody that is specific for the RSV G ectodomain protein in thepre-immune subject prior to the administration step. In a furtherembodiment, the single administration of the ND VLP that comprises theRSV G ectodomain protein to the first pre-immune mammalian subjectincreases the level of antibody that is specific for the RSV Gectodomain protein compared to the level of the antibody in a secondimmunized subject, wherein the second immunized subject is a secondpre-immune subject that is immunized with a second compositioncomprising chimeric ND VLPs that contain, in operable combination 1)stabilized post-fusion RSV F protein ectodomain or stabilized pre-fusionRSV F protein ectodomain, 2) TM domain of NDV F protein, 3) CT domain ofNDV F protein, 4) CT domain of NDV HN protein, 5) TM domain of NDV HNprotein, and 6) RSV G ectodomain protein. In a particular embodiment,lung tissue of the first immunized subject contains a lower RSV titerthan lung tissue of a naïve subject to which the first composition hasnot been administered. In yet another embodiment, the level of the RSVneutralizing antibodies after a single administration of the firstcomposition to the first pre-immune subject is higher than the level ofRSV neutralizing antibodies after a single administration of the firstcomposition to a naïve subject. In an additional embodiment, the levelof the RSV neutralizing antibodies after a single administration of thefirst composition to the first pre-immune subject is higher for at leasta period from about 30 to about 220 days after the single administrationthan the level of RSV neutralizing antibodies after a singleadministration of the second composition to the second pre-immunesubject. In a further embodiment, the level of splenic memory B cells inthe first immunized mammalian subject is higher than the level ofsplenic memory B cells in the second immunized subject. In anotherembodiment, the level of splenic memory B cells in the first immunizedmammalian subject is higher than the level of splenic memory B cellsafter RSV infection of the first pre-immune subject. In a furtherembodiment, the level of splenic memory B cells in the first immunizedmammalian subject is higher than the level of splenic memory B cellsafter RSV infection of a naïve subject. In another embodiment, avidityof the RSV antibodies in the first immunized mammalian subject is higherthan avidity of RSV antibodies in a pre-immune mammalian subjectinfected with RSV. In a further embodiment, the avidity of the antibodythat is specific for the RSV G ectodomain protein in the first immunizedmammalian subject is higher than avidity of antibody that is specificfor the RSV G ectodomain protein in a pre-immune mammalian subjectinfected with RSV.

The invention also provides a vaccine comprising recombinant chimericNewcastle Disease virus-like particles (ND VLPs) that contain a chimericprotein comprising, in operable combination, 1) stabilized pre-fusionRSV F protein ectodomain, 2) transmembrane (TM) domain of NDV F protein,and 3) cytoplasmic (CT) domain of NDV F protein. In a particularembodiment, the vaccine further comprises, in operable combination,foldon sequence listed as SEQ ID NO:14 and/or RSV G ectodomain proteinsequence. In a particular embodiment, the RSV G ectodomain proteinsequence is operably linked to NDV HN TM domain and to NDV FIN CTdomain.

The invention contemplates combining and/or removing and/or substitutingfeatures from different embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B: Immunization of pre-immune mice vs naïve mice. Left panelFIG. 1A: neutralization titers (NA) in mice previously infected (primed)with RSV and then immunized with RSV, Pre-F VLPs, or Post-F VLPs(boost). NA titers in pooled sera at each time point were determined byplaque reduction assays in Hep-2 cells. Right panel FIG. 1B: NA titersfrom naïve mice primed and then boosted (day 90) with Pre-F or Post-FVLPs or RSV infected. Mock: infected/immunized with buffer. Titers on alog scale.

FIG. 2: Incorporation of RSV F Ectodomain into ND VLPs by to constructan “F/F chimera protein” exemplified by fusing RSV F ectodomain to theTM and CT domains of NDV F protein.

FIG. 3: Alterations of the RSV F Protein Ectodomain (McLellan, et alScience 340:1113 (2013), Swanson et al PNAS 108:9619 (2011)).

FIG. 4: Test of immunogenicity of VLPs as a vaccine in an animal(exemplified by mouse).

FIG. 5A-B: DNA sequence (SEQ ID NO:01) (FIG. 5A) and encoded proteinsequence (FIG. 5B) (SEQ ID NO:02) of the chimeric “RSV pre-F/F protein,”containing stabilized pre-fusion RSV F protein ectodomain (19) (SEQ IDNOs:05-06) (italics), transmembrane (TM) domain of NDV F protein (SEQ IDNOs:09-10) (underlined), cytoplasmic (CT) domain of NDV F protein (SEQID NOs:11-12) (lower case), and foldon sequence (SEQ ID NOs:13-14)(bold).

FIG. 6A-B: : DNA sequence (FIG. 6A) (SEQ ID NO:03) and encoded proteinsequence (FIG. 6B) (SEQ ID NO:04) of the chimeric “RSV post-F/F protein”containing stabilized post-fusion RSV F protein ectodomain (19) (SEQ IDNOs:07-08) (italics), transmembrane (TM) domain of NDV F protein (SEQ IDNOs:09-10) (underlined), and cytoplasmic (CT) domain of NDV F protein(SEQ ID NOs:11-12) (lower case).

FIG. 7A-B: Stabilized pre-fusion RSV F protein ectodomain (19): (FIG.7A) DNA sequence (SEQ ID NO:05), and (FIG. 7B) encoded protein sequence(SEQ ID NO:06).

FIG. 8A-B: Stabilized post-fusion RSV F protein ectodomain (19): (FIG.8A) DNA sequence (SEQ ID NO:07), and (FIG. 8B) encoded protein sequence(SEQ ID NO:08).

FIG. 9A-C: DNA sequences and encoded protein sequences of (FIG. 9A)transmembrane (TM) domain of NDV F protein (SEQ ID NOs:09-10), (FIG. 9B)cytoplasmic (CT) domain of NDV F protein (SEQ ID NOs:11-12), and foldonsequence (SEQ ID NOs:13-14).

FIG. 10A-D: Protein Content of VLPs. Panel FIG. 10A shows a Western blotof proteins present in stocks of VLP-H/G+Pre-F/F and VLP-H/G+Post-F/F.Proteins (electrophesed in the presence of reducing agent) in apolyacrylamide gel containing duplicate lanes of the proteins in the twoVLPs were transferred to a membrane. One half was incubated with anti-Fantibody (lanes M, 1, 2). The other half was incubated with anti-Gprotein antibody (lanes 3, 4). M: marker Pre-F/F protein. Lanes 1, 3:VLP-H/G+Pre-F/F; Lanes 2, 4: VLP-H/G+Post-F/F. The panel shows resultsof one of 3 separate blots with identical results. Panels B and C showbinding of different concentrations of mAb motivizumab (panel FIG. 10B)or mAb D25 (Panel FIG. 10C) to each VLP in an ELISA as previouslydescribed²¹. Panel FIG. 10D shows binding of an anti-G protein antibodyto two different concentrations of VLPs (concentrations in ng of Fprotein). Results were identical in three or four separatedeterminations.

FIG. 11A-B: Immunization/Infection Timelines. Panel FIG. 11A showstiming of infection of animals with RSV (day 0) and their subsequentimmunization with VLPs (day 95) or a second RSV infection (day 95). Serawere harvested from each animal at times indicated by arrows pointingupwards. Panel FIG. 11B shows timing of prime immunization with VLPs orRSV infection of naïve animals (day 0) and the subsequent boost withVLPs or a second RSV infection (day 100). Sera were harvested at timesindicated by arrows pointing upward. In one embodiment, blood collectionwas extended from 128 to 250 days (RSV primed) or 200 days (naïve) inorder to to determine durability of serum antibodies and monitor memoryat later times after immunization.

FIG. 12A-B: Neutralization Titers in Sera from RSV-experienced or NaïveAnimals. Panel FIG. 12A shows neutralization titers in pooled sera aftera single immunization with VLPs of RSV previously infected animals. Atday 128, the difference between results of VLP-H/G+Pre-F/F immunizationand VLP-H/G+Post-F/F immunization was significant with a p value of0.0009. The difference between VLP-H/G+Pre-F/F and RSV immunization wassignificant with a p value of 0.0005. Difference betweenVLP-H/G+Post-F/F and RSV immunization was not significant. All resultsare the average of four separate determinations with mean and standarddeviation shown. Panel FIG. 12B shows neutralization titers in pooledsera after a prime and after a boost of naïve animals with VLPs or RSV.At day 71, p values for the difference between results of immunizationwith VLP-H/G+Pre-F/F and VLP-H/G+Post-F/F was 0.0005 and for thedifference between VLP-H/G+Pre-F/F and RSV was 0.0030. The differencebetween VLP-H/G+Post-F/F and RSV was not significant. At day 128, thedifference between results of VLP-H/G+Pre-F/F immunization andVLP-H/G+Post-F/F immunization was not significant. The p values fordifference between VLP-H/G+Pre-F/F and RSV immunization was 0.035 andfor the difference between VLP-H/G+Post-F/F and RSV immunization was0.0012. Results are the average of three separate determinations withmean and standard deviation shown.

FIG. 13A-D: Total Anti-F Protein Antibody in Animal Sera. Total anti-Fprotein antibody was measured in ELISA using as target purified solublepre-fusion F (panels FIGS. 13A and C) or purified soluble post-fusion Fprotein (panels FIG. 13B and D). Panels FIGS. 13A and B show ng/ml ofanti-F protein IgG at different time points in RSV-experienced animals.Results are the average of two separate determinations. For the pre-Ftarget as well as post-F target the difference at day 128 betweenRSV/VLP-H/G+Pre-F/F and RSV/VLP-H/G+Post-F/F groups was not significant.For the pre-F target, p value for difference between RSV/VLP-H/G+Pre-F/Fand RSV/RSV was 0.030 while the difference between RSV/RSV andRSV/VLP-H/G+Post-F/F immunization was not significant. For the post Ftarget, the p values for differences between RSV/VLP-H/G+Pre-F/F orRSV/VLP-H/G+Post-F/F VLP immunization and RSV/RSV immunization were0.034 and 0.0011, respectively. Panels FIGS. 13C and D show ng/ml ofanti-F protein IgG at different time-points in immunized naïve animals.Figure shows results of one of two determinations with identical resultsand replicates results previously reported²¹. For the pre-F target orthe post-F target the differences in values at day 128 between allgroups were not significant.

FIG. 14A-B: Total Anti-G Protein Antibody in Animal Sera. Total anti-Gprotein IgG was measured in ELISA using as target soluble G protein.Panel FIG. 14A shows ng/ml of anti-G protein IgG at different times inRSV-experienced animals. The results are the average of four separatedeterminations with average and standard deviations shown. At day 128, pvalue for the difference between RSV/VLP-H/G+Pre-F/F andRSV/VLP-H/G+Post-F/F immunization was 0.0057, the p value for thedifference between RSVNLP-H/G+Post-F/F and RSV/RSV was 0.002. The pvalue for the difference between RSV/VLP-H/G+Pre-F/F and RSV/RSV was0.0002. Panel FIG. 14B shows ng/ml of anti-G protein IgG in immunizednaïve animals. Results are the average of two separate determinationswith standard deviations shown. At day 128, p values for differencesbetween VLP-H/G+Pre-F/F and VLP-H/G+Post-F/F immunization, for RSV andVLP-H/G+Pre-F/F-VLP immunization, and for RSV and VLP-H/G+Post-F/Fimmunization were 0.042, 0.019, and 0.055 respectively.

FIG. 15: Protection from Challenge. Shown are lung titers afterchallenge of RSV-experienced VLP immunized animals. RSV challenge was125 days after VLP immunization. A: no RSV prime, no immunization; B:RSV primed, RSV immunized; C: RSV primed, VLP-H-G+Pre-F/F immunized; D:RSV primed, VLP-H/G+Post-F/F immunized. Each group contained fiveanimals and titers of each animal are shown in the graph. The p valuefor the differences between group A and the other groups is 0.0182.

FIG. 16A-B: Levels and Durability of Neutralizing Antibody Titers. PanelFIG. 16A shows neutralization titers in RSV primed animals. Panel FIG.16B shows neutralization titers in Naïve animals.

FIG. 17A-D: Total anti-F IgG titers in (FIG. 17A) pre-F protein targetRSV primed mice, (FIG. 17B) post-F protein target RSV primed mice, (FIG.17C) pre-F protein target naïve mice, and (FIG. 17D) post-F proteintarget naïve mice. Circles denote RSV/Pre-F/F VLPs (panels FIGS. 17A andB), squares denote RSV/Post-F/F VLPs (Panels FIGS. 17A and B), andtriangles denote RSV/RSV (Panels FIG. 17A, B, C, D); circles denotePre-F/F VLPs/Pre-F/F VLPs, squares denote Post-F/F VLPs/Post F/F VLPs(Panels FIGS. 17C and D).

FIG. 18A-C: Total anti-G protein IgG Titers in (FIG. 18A) RSV primedmice, and (FIG. 18B) naïve mice. Panel FIG. 18C is the same as panel Bfor naïve mice, except with the Y axis scale is changed.

FIG. 19A-B: Anti-pre-F protein antibody secreting splenic B cells in(FIG. 19A) RSV primed animals, and (FIG. 19B) naïve animals. Circlesdenote RSV/Pre-F/F VLPs (Panel FIG. 19A) or Pre-F/F VLP/Pre-F/F VLPs(Panel FIG. 19B), squares denote RSV/Post-F/F VLPs (panel FIG. 19A) orPost-F/F VLPs/Post-F/F VLPs (panel FIG. 19B), and triangles denoteRSV/RSV.

FIG. 20A-B: anti-post-F protein antibody secreting splenic B cells in(FIG. 20A) RSV primed animals, and (FIG. 20B) naïve animals. Circlesdenote RSV/Pre-F/F VLPs, squares denote RSV/Post-F/F VLPs, and trianglesdenote RSV/RSV.

FIG. 21A-C: Measure of Avidity/Stability of Antigen-antibody Complexesin Increasing Urea for (FIG. 21A) pre-F protein target, (FIG. 21B)post-F protein target, and (FIG. 21C) G protein target.

FIG. 22A-D shows avidity of F protein antibodies stimulated in (FIG.22A) RSV primed animals with pre-F protein target, (FIG. 22B) RSV primedanimals with post-F protein target, (FIG. 22C) Naïve animals with pre-Fprotein target, and (FIG. 22D) Naïve animals with post-F protein target.

FIG. 23A-B shows avidity of G protein antibodies stimulated in (FIG.23A) RSV primed animals, and (FIG. 23B) Naïve animals.

FIG. 24A-D shows (FIG. 24A) NDV HN/RSV G (H/G) DNA Sequence (SEQ IDNO:15), (FIG. 24B) NDV HN/RSV G (H/G)Amino Acid Sequence (SEQ ID NO:16),(FIG. 24C) RSV G DNA Sequence (SEQ ID NO:17) (GenBank No. X73355), and(FIG. 24D) RSV G Amino Acid Sequence (SEQ ID NO:18) (GenBank No.X73355). NDV HN Cytoplasmic Tail is in bold text; NDV HN TransmembraneRegion is in underlined bold text; RSV G Ectodomain is in italics text;RSV G Cytoplasmic Tail is in underlined italics text; and RSV GTransmembrane Region is in bold italics text.

DEFINITIONS

To facilitate understanding of the invention, a number of terms aredefined below. The term “recombinant” molecule refers to a molecule thatis produced using molecular biological techniques. Thus, “recombinantDNA molecule” refers to a DNA molecule that is comprised of segments ofDNA joined together by means of molecular biological techniques. A“recombinant protein” or “recombinant polypeptide” as used herein refersto a protein molecule that is expressed using a recombinant DNAmolecule. A “recombinant” virus-like particle (VLP) refers to a VLP thatis expressed using a recombinant DNA molecule.

“Operable combination” and “operably linked” when in reference to therelationship between nucleic acid sequences and/or amino acid sequencesrefers to linking (i.e., fusing) the sequences in frame such that theyperform their intended function. For example, when linking RSV F proteinectodomain to the TM domain of NDV F protein and to the CT domain of NDVF protein for the purpose of producing a VLP that elicits RSVneutralizing antibodies, then the arrangement and orientation of thelinked sequences is such that a VLP is formed, and the RSV F protein isexpressed as part of the VLP to elicit RSV neutralizing antibodies. Thisis exemplified in the constructs of FIGS. 2, 5A-B, and 6A-B. In anotherexample, operably linking a promoter sequence to a nucleotide sequenceof interest refers to linking the promoter sequence and the nucleotidesequence of interest in a manner such that the promoter sequence iscapable of directing the transcription of the nucleotide sequence ofinterest and/or the synthesis of a polypeptide encoded by the nucleotidesequence of interest.

The term “matrix protein,” “membrane protein”, or “M protein” as usedherein, means any protein localized between the envelope and thenucleocapsid core and facilitates the organization and maintenance ofthe virion structure and budding processes. Exemplary NDV M proteinsequences include those described in U.S. Pat. No. 7,951,384 issued toMorrison et al. on May 1, 2011; U.S. Pat. No. 8,974,797, issued toMorrison on Mar-10-2015, each of which is incorporated by reference.

The term “nucleocapsid protein” or “NP protein” as used herein, meansany protein that associates with genomic RNA (i.e., for example, onemolecule per hexamer) and protects the RNA from nuclease digestion.Exemplary NP protein sequences from NDV include those described in U.S.Pat. No. 7,951,384 issued to Morrison et al. on May 1, 2011; U.S. Pat.No. 8,974,797, issued to Morrison on Mar. 10, 2015, each of which isincorporated by reference.

The term “fusion protein” or “F protein” as used herein, means anyprotein that projects from the envelope surface and mediates host cellentry by inducing fusion between the viral envelope and the cellmembrane. However, it is not intended that the present invention belimited to functional F proteins. For example, an F protein may beencoded by a mutant F gene such as, but not limited to, F-K115Q. F-K115Qis believed to eliminate the normal cleavage and subsequent activationof the fusion protein. F-K115Q mimics naturally occurring F-proteinmutations in avirulent NDV strains, and in cell culture, eliminates anypotential side effects of cell-cell fusion on the release of VLPs.Exemplary NDV F protein sequences include those described in U.S. Pat.No. 7,951,384 issued to Morrison et al. on May 1, 2011; U.S. Pat. No.8,974,797, issued to Morrison on Mar. 10, 2015, each of which isincorporated by reference.

The term “haemagglutinin-neuraminidase protein”, “HN protein”, or Gprotein as used herein, means any protein that spans the viral envelopeand projects from the surface as spikes to facilitate cell attachmentand entry (i.e., for example, by binding to sialic acid on a cellsurface). These proteins possess both haemagglutination andneuraminidase activity. Exemplary NDV HN protein sequences include thosedescribed in U.S. Pat. No. 7,951,384 issued to Morrison et al. on May 1,2011; U.S. Pat. No. 8,974,797, issued to Morrison on Mar. 10, 2015, eachof which is incorporated by reference.

The term Respiratory Syncytial Virus (RSV) “G protein sequence” refersto the RSV attachment glycoprotein, and is exemplified by the amino acidsequence (SEQ ID NO:18) (GenBank No. X73355 for RSV strain A) (FIG.24D), that is encoded by the RSV G DNA sequence (SEQ ID NO:17) (FIG.24C).

A “recombinant” sequence (such as a DNA sequence, RNA sequence, andprotein sequence) refers to a DNA sequence, RNA sequence, and proteinsequence, respectively that is comprised of segments of a DNA, RNA, andprotein that are joined together by means of molecular biologicaltechniques.

A “chimeric” polypeptide refers to a polypeptide that contains at leasttwo amino acid sequences that are covalently linked together to create acombination of sequences that does not exist in nature. The two aminoacid sequences may be derived from different sources (e.g., differentorganisms, different tissues, different cells, etc.) and/or may bedifferent sequences from the same source and/or may include wild typeand mutant sequences. In one embodiment, the chimeric polypeptide is arecombinant polypeptide containing sequences from two different viruses,such as from Newcastle disease virus (NDV) and Respiratory SyncytialVirus (RSV).

“F/F chimera protein” and “F/F protein” interchangeably refer to aprotein in which a fusion (F) protein ectodomain is linked to the TM andCT domains of NDV F protein as shown in FIG. 2. The linked F proteinectodomain may be the wild type RSV F protein ectodomain to generate achimeric “RSV F/F protein” (U.S. Pat. No. 8,580,270, issued to MorrisonNov. 12, 2013; U.S. Pat. No. 9,168,294, issued to Morrison Oct. 27,2015). Alternatively, the linked F protein ectodomain may be thestabilized pre-fusion RSV F protein ectodomain (described in 19)(exemplified by SEQ ID NO:05, FIG. 7A) to generate a chimeric “RSVpre-F/F protein,” exemplified by SEQ ID NO:02 (FIG. 5B), which containsthe optional foldon sequence SEQ ID NO:14 (FIG. 9C). In yet anotheralternative, the linked F protein ectodomain may be the stabilizedpost-fusion RSV F protein ectodomain (described in 19) (exemplified bySEQ ID NO:08, FIG. 8B) to generate a chimeric “RSV post-F/F protein”exemplified by SEQ ID NO:04, FIG. 6B.

“Newcastle Disease Virus” and “NDV” refer to a negative-sensesingle-stranded RNA virus of the family Paramyxoviridae that causes ahighly contagious zoonotic bird disease affecting many domestic and wildavian species.

“Respiratory Syncytial Virus” and “RSV” refer to a negative-sense,single-stranded RNA virus of the family Paramyxoviridae that causes arespiratory disease, especially in children. RSV is a member of theparamyxovirus subfamily Pneumovirinae. Its name comes from the fact thatF proteins on the surface of the virus cause the cell membranes onnearby cells to merge, forming syncytia.

The terms “virus-like particle” and “VLP” as used herein, refer to anon-infective viral subunit that contains viral proteins that form avirus's outer shell and the surface proteins, resembles the externalconformation of the virus from which the VLP was derived, and lacksviral DNA or RNA genome. A VLP comprising viral capsid proteins mayundergo spontaneous self-assembly. In some embodiments, these viralproteins are embedded within a lipid bilayer. In some embodiments a“VLP”” interchangeably refer to a non-replicating, non-infectiousparticle shell that contains one or more virus proteins, lacks the viralRNA and/or DNA genome, and that approximately resembles live virus inexternal conformation. Methods for producing and characterizingrecombinant VLPs containing Newcastle Disease Virus (NDV) proteins havebeen described (Pantua et al. (2006) J. Virol. 80:11062-11073; U.S. Pat.No. 7,951,384 issued to Morrison et al. on May 1, 2011; U.S. Pat. No.8,974,797, issued to Morrison on Mar. 10, 2015, each of which isincorporated by reference). Further methods for producing NDV VLPs aredisclosed herein.

“ND VLP” and “Newcastle Disease virus like particle” interchangeablyrefer to a VLP containing at least one Newcastle Disease Virus protein,preferably, at least NDV “matrix protein” (also referred to as “membraneprotein” and “M protein”), which is a NDV protein localized between theenvelope and the nucleocapsid core and facilitates the organization andmaintenance of the virion structure and budding processes (U.S. Pat. No.8,974,797, issued to Morrison on Mar. 10, 2015). Methods for producing,characterizing, and purifying recombinantly produced ND VLPs are knownin the art (Pantua et al. (2006) J. Virol. 80:11062-11073; U.S. Pat. No.8,974,797, issued to Morrison on Mar. 10, 2015; U.S. Pat. No. 9,168,294issued to Morrison on Oct. 27, 2015).

“Pre-F/F VLPs” refer to ND VLPs that contain a stabilized pre-fusion RSVF protein ectodomain.

“Post-F/F VLPs” refer to ND VLPs that contain a stabilized post-fusionRSV F protein ectodomain.

“Symptoms of RSV infection” include runny nose, decrease in appetite,coughing, sneezing, fever, wheezing, irritability, decreased activity,breathing difficulties, pneumonia, bronchiolitis, morbidity, andmortality.

The term “ectodomain” when in reference to a membrane protein refers tothe portion of the protein that is exposed on the extracellular side ofa lipid bilayer of a cell, virus and the like.

“Wild type RSV F protein ectodomain” is exemplified by the sequencedescribed in Tale 7 and FIGS. 219-220 of U.S. Pat. No. 8,974,797, issuedto Morrison on Mar. 10, 2015.

“Stabilized pre-fusion RSV F protein ectodomain is described in McLellanet al. (19) and exemplified by SEQ ID NO:06; FIG. 7B.

“Stabilized post-fusion RSV F protein ectodomain (described in McLellanet al. (38) and exemplified by SEQ ID NO:08; FIG. 8B.

“Transmembrane domain of NDV F protein” and “TM domain of NDV F protein”interchangeably refer to a protein sequence, and portions thereof, thatspans the lipid bilayer of NDV. The TM domain of NDV F protein isexemplified by SEQ ID NO:10; FIG. 9A, and by Table 4 of U.S. Pat. No.8,974,797, issued to Morrison on Mar. 10, 2015.

“Cytoplasmic domain of NDV F protein” and “CT domain of NDV F protein”interchangeably refer to refer to a protein sequence, and portionsthereof, that is on the cytoplasmic side or virion interior or VLPinterior of the lipid bilayer of NDV. The CT domain of NDV F protein isexemplified by SEQ ID NO:12; FIG. 9B, and by Table 4 of U.S. Pat. No.8,974,797, issued to Morrison on Mar. 10, 2015.

The term “Pre-F/F VLP” herein refers to VLP-H/G+Pre-F/F; “Post-F/F VLPs”refers to VLP-H/G+Post-F/F VLPs.

The term “H/G” herein refers to the RSV G ectodomain, operably linked toCT of NDV HN and to TM of NDV HN.

The term “RSV G ectodomain” protein is exemplified by the sequence shownin italics text of the RSV G Amino Acid Sequence (SEQ ID NO:18) (GenBankNo. X73355 for RSV A Strain) of FIG. 24D, which is encoded by the DNAsequence shown in italics text of RSV G DNA Sequence (SEQ ID NO:17)(GenBank No. X73355 for RSV A Strain) of FIG. 24C.

The term “expression vector” refers to a nucleotide sequence containinga desired coding sequence and appropriate nucleic acid sequencesnecessary for the expression (i.e., transcription into RNA and/ortranslation into a polypeptide) of the operably linked coding sequencein a particular host cell. Expression vectors are exemplified by, butnot limited to, plasmid, phagemid, shuttle vector, cosmid, virus,chromosome, mitochondrial DNA, plastid DNA, and nucleic acid fragmentsthereof.

Nucleic acid sequences used for expression in prokaryotes include apromoter, optionally an operator sequence, a ribosome binding site andpossibly other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals.

“Mammalian subject” includes human, non-human primate, murine (e.g.,mouse, rat, cotton rat), ovine, bovine, ruminant, lagomorph, porcine,caprine, equine, canine, felines, ave, etc.

A subject “in need” of reducing one or more symptoms of a disease,and/or “in need” for a particular treatment (such as immunization)against a disease includes a subject that exhibits and/or is at risk ofexhibiting one or more symptoms of the disease. For example, a subjectmay be in need of a reduction in one or more of (a) level of RSVinfection, (b) one or more symptoms of RSV infection, (c) susceptibilityto RSV infection, and (d) transmission of RSV infection, compared to afirst pre-immune subject. In another example, subjects may be at riskbased on family history, genetic factors, environmental factors, etc.This term includes animal models of the disease. Thus, administering acomposition (which reduces a disease and/or which reduces one or moresymptoms of a disease) to a subject in need of reducing the diseaseand/or of reducing one or more symptoms of the disease includesprophylactic administration of the composition (i.e., before the diseaseand/or one or more symptoms of the disease are detectable) and/ortherapeutic administration of the composition (i.e., after the diseaseand/or one or more symptoms of the disease are detectable). Theinvention's compositions and methods are also useful for a subject “atrisk” for disease refers to a subject that is predisposed to contractingand/or expressing one or more symptoms of the disease. Thispredisposition may be genetic (e.g., a particular genetic tendency toexpressing one or more symptoms of the disease, such as heritabledisorders, etc.), or due to other factors (e.g., environmentalconditions, exposures to detrimental compounds, including carcinogens,present in the environment, etc.). The term subject “at risk” includessubjects “suffering from disease,” i.e., a subject that is experiencingone or more symptoms of the disease. It is not intended that the presentinvention be limited to any particular signs or symptoms. Thus, it isintended that the present invention encompass subjects that areexperiencing any range of disease, from sub-clinical symptoms tofull-blown disease, wherein the subject exhibits at least one of theindicia (e.g., signs and symptoms) associated with the disease.

The terms “Pre-immune,” “RSV primed,” and “RSV experienced” subject areused interchangeably to refer to a subject that has previously been incontact with, and/or previously infected by, RSV and/or an antigenicportion of RSV. This is exemplified by a subject that has beenvaccinated against RSV through active human intervention, and/or asubject that has been exposed to RSV in the environment (e.g., bycontact with another subject that is infected with RSV and/or contactwith body fluid or sample from another subject that is infected withRSV). In one embodiment, a pre-immune subject contains detectable levelsof RSV neutralizing antibodies (FIG. 1A, and FIG. 12A), albeit theselevels are not sufficient to (a) reduce (including prevent) subsequentRSV infection, (b) reduce symptoms of RSV infection, (c) reducesusceptibility to RSV infection, and/or (d) reduce transmission of RSVinfection from the pre-immune subject.

“Naïve subject” refers to a subject that has not been previously incontact with, and/or previously infected by, RSV and/or an antigenicportion of RSV. In one embodiment, a naïve subject does not containdetectable levels of RSV neutralizing antibodies (FIG. 1B, and FIG.12B).

“Immunogenically effective amount” refers to that amount of a moleculethat elicits and/or increases production of neutralizing antibody in ahost upon vaccination with the molecule.

The term “vaccine” refers to a pharmaceutically acceptable preparationthat may be administered to a host to induce a humoral immune response(including eliciting a soluble antibody response), and/or cell-mediatedimmune response (including eliciting a cytotoxic T lymphocyte (CTL)response). The dosage of the pharmaceutical formulation can bedetermined readily by the skilled artisan, for example, by firstidentifying doses effective to elicit a prophylactic or therapeuticimmune response, e.g., by measuring the serum titer of virus specificimmunoglobulins or by measuring the inhibitory ratio of antibodies inserum samples, or urine samples, or mucosal secretions. Said dosages canbe determined from animal studies. Vaccines may contain pharmaceuticallyacceptable carriers, adjuvants, and/or excipients. “Carriers” and“diluents” include water, saline solution, human serum albumin, oils,polyethylene glycols, aqueous dextrose, glycerin, propylene glycol orother synthetic solvents. “Adjuvant” refers to any compound which, wheninjected together with an antigen, non-specifically enhances the immuneresponse to that antigen. “Excipient” is an inactive substance used as acarrier for the invention's compositions that may be useful fordelivery, absorption, bulking up to allow for convenient and accuratedosage of the invention's compositions.

The term “administering” to a subject means delivering a molecule to asubject. “Administering” a composition to a subject in need ofimmunization against virus infection and/or in need of reducing one ormore disease symptoms, includes prophylactic administration of thecomposition (i.e., before virus infection and/or before one or moresymptoms of the disease are detectable) and/or therapeuticadministration of the composition (i.e., after virus infection and/orafter one or more symptoms of the disease are detectable).Administration also may be concomitant with (i.e., at the same time as,or during) virus infection and/or detection of one or more diseasesymptoms.

Methods of administering the invention's compositions include, withoutlimitation, administration in intranasal, parenteral, intraperitoneal,sublingual forms. In one preferred embodiment, administration isintranasal.

“Neutralizing antibody” (“NA”) refers to an antibody that specificallybinds to a target antigen, and neutralizes (i.e., reduces) one or moreof the biological effects and/or functions of the antigen. This givesneutralizing antibodies the ability to fight viruses which attack theimmune system, since they can neutralize (i.e., reduce) virus function(e.g., by reducing virion binding to receptors, reducing virus uptakeinto cells, reducing uncoating of the viral genomes in endosomes, and/orcausing aggregation of virus particles. This is in contrast tonon-neutralizing antibodies that are also produced after viralinfection, and that bind specifically to virus particles, but do notneutralize (i.e., reduce) one or more of the biological effects and/orfunctions of the antigen to which they bind. Thus, neutralizingantibodies can produce reduced infectivity by the virus, reduced viruspathology (VEP), reduced susceptibility to infection (VES) by the virus,and/or reduced transmission of the virus. Methods for determining thepresence and level of RSV neutralizing antibodies and nonneutralizingantibodies are known in the art (19, 30, 36), including a classical invitro plaque reduction assay (36). Briefly, in this assay, pooled seraare used as are sera from individual subjects. For kinetics of inductionof neutralizing antibodies, equal aliquots of sera from animals in eachgroup obtained at multiple (e.g., four) times after immunization arepooled and used to determine titers. Virus titers of a stock of virusobtained after mixing virus with an equivalent volume of sera frombuffer immunized subjects (TNE) are taken as 100% to account for anynonspecific neutralization by sera (control). The dilution of seraresulting in reduction in titer by 50% of the control is used todetermine neutralization titers. Thus, a “detectable” level of RSVneutralizing antibody titer is a dilution of sera greater than 2, asexemplified by naïve subjects primed and then boosted (day 90) withPre-F or Post-F VLPs or RSV infected (FIG. 1A, and FIG. 12A).Conversely, an “undetectable” level of RSV neutralizing antibody titeris a dilution of sera of less than 2, as exemplified by the naïve mocksubjects that are infected/immunized with buffer (FIG. 1A).

“Nonneutralizing antibody” refers to an antibody that specifically bindsto a target antigen, and does not neutralize (i.e., does not reduce) oneor more of the biological effects and/or functions of the antigen. Mostof the antibody made after RSV infection is nonneutralizing. Thisnonneutralizing antibody binds to virus but does not reduce RSVinfection. This nonneutralizing antibody could inhibit induction ofneutralizing antibody after RSV vaccination.

The terms “anti-G protein antibody” and “antibody that is specific forthe RSV G protein” are interchangeably used to refer to an antibody(such as immunoglobulin G (IgG)) that specifically binds to RSV Gprotein and/or to antigenic portions thereof.

The terms “anti-F protein antibody” and “antibody that is specific forthe RSV F protein” are interchangeably used to refer to an antibody(such as immunoglobulin G (IgG)) that specifically binds to RSV Fprotein and/or to antigenic portions thereof.

The terms “specific,” “specifically binds,” and grammatical equivalents,when made in reference to the binding of antibody to a molecule (e.g.,binding of IgG to stabilized pre-fusion RSV F protein ectodomain F)refers to an interaction of the antibody with one or more epitopes onthe molecule where the interaction is dependent upon the presence of aparticular structure on the molecule.

The term “durable” and grammatical equivalents, when referring toantibody (such as neutralizing antibody) means that the detectablelevels of antibody continue to be present in serum over a period oftime. Durability also refers to the persistence of serum antibodiesafter immunization. Levels of serum antibodies (e.g., at 50-100 daysafter immunization) may be determined by measuring the levels of longlived, bone marrow associated plasma cells (LLPCs). That is, antibodydurability is a measure of the levels of LLPCs.

The terms “avidity” and “affinity” when in reference to the interactionbetween an antibody and an antigen interchangeably refer to the strengthof binding of the antibody to the antigen. Avidity Affinity isdetermined by on rates and off rates of antibody binding to antigen. Bcells undergo affinity maturation in germinal centers. With increasingtime, B cells secreting higher affinity antibodies are selected andpersist while B cells secreting lower affinity antibodies areeliminated. Because polyclonal antibodies are a mixture of antibodieswith different binding sites on an antigen and different affinities forthat antigen, on and off rates cannot be precisely determined. Thus thestrength of binding of polyclonal antibodies to an antigen is measuredby the avidity of the antibodies. Avidity is classically defined andmeasured by the stability of the binding of polyclonal antibodies to anantigen in the presence of increasing concentrations of urea, rangingfrom 1-7 M. The results are plotted as the percent of binding atdifferent urea concentrations with 100% representing the binding in nourea. The results are the average of all the antibodies in thepopulation of antibodies in the serum. Lower percentages indicate weakerbinding or weaker avidity. Methods for measuring avidity are known inthe art (Delgado et al. 2009. Nature Med. 15:34-41, and Polack et al.2003. Nat Med 9:1209-1213).

The term “splenic memory B cells” refer to long-lived B lymphocytes thatare generated in the spleen. When an animal is immunized (or infectedwith a pathogen), the adaptive, T cell dependent immune response resultsin plasmablasts followed by the formation of germinal centers (3-4 daysafter antigen exposure) in the spleen. Germinal centers are the site inthe spleen where memory B cells and LLPC (long lived plasma cells) aregenerated. Memory B cells form first followed by LLPC. Weise et al. 2016Immunity 44: 116-130 estimates that memory B cells form beginning at 6-8days after immunization with the formation rate peaking at day 11-26.Numbers continue to accumulate at least 40 days. In contrast, the rateof formation of LLPCs peaks at 30-32 days. LLPCs migrate to the bonemarrow and generally persistently secrete antibody. They are largelyresponsible for the levels of serum antibody detected in unstimulatedanimals for prolonged periods of time (months, years, life of animal)with the duration likely depending upon the antigen. Memory B cells,after antibody class switching and affinity maturation, remain quiescentin the spleen until a second exposure to the antigen (infection orvaccine), at which time they are activated and begin secreting antibody.Memory B cells are a primary defense against infections that animalshave previously experienced.

The terms “increase,” “elevate,” “raise,” and grammatical equivalents(including “higher,” “greater,” etc.) when in reference to the level ofany molecule (e.g., amino acid sequence, and nucleic acid sequence,antibody (such as IgG, anti-F protein antibody, and anti-G proteinantibody), etc.), cells (such as memory B cells), and/or phenomenon(e.g., the level of RSV neutralizing antibodies, the titer RSVneutralizing antibodies, avidity, disease symptom, virus function,virion binding to receptors, virus uptake into cells, uncoating of theviral genomes in endosomes, infectivity by the virus, virus pathology(VEP), susceptibility to infection (VES) by the virus, transmission ofthe virus, etc.) in a first sample (or in a first subject) relative to asecond sample (or relative to a second subject), mean that the quantityof the molecule, cell and/or phenomenon in the first sample (or in thefirst subject) is higher than in the second sample (or in the secondsubject) by any amount that is statistically significant using anyart-accepted statistical method of analysis. In one embodiment, thequantity of the molecule, cell and/or phenomenon in the first sample (orin the first subject) is at least 10% greater than, at least 25% greaterthan, at least 50% greater than, at least 75% greater than, and/or atleast 90% greater than the quantity of the same molecule, cell and/orphenomenon in the second sample (or in the second subject). Thisincludes, without limitation, a quantity of molecule, cell, and/orphenomenon in the first sample (or in the first subject) that is atleast 10% greater than, at least 15% greater than, at least 20% greaterthan, at least 25% greater than, at least 30% greater than, at least 35%greater than, at least 40% greater than, at least 45% greater than, atleast 50% greater than, at least 55% greater than, at least 60% greaterthan, at least 65% greater than, at least 70% greater than, at least 75%greater than, at least 80% greater than, at least 85% greater than, atleast 90% greater than, and/or at least 95% greater than the quantity ofthe same molecule, cell and/or phenomenon in the second sample (or inthe second subject). In one embodiment, the first sample (or the firstsubject) is exemplified by, but not limited to, a sample (or subject)that has been manipulated using the invention's compositions and/ormethods. In a further embodiment, the second sample (or the secondsubject) is exemplified by, but not limited to, a sample (or subject)that has not been manipulated using the invention's compositions and/ormethods. In an alternative embodiment, the second sample (or the secondsubject) is exemplified by, but not limited to, a sample (or subject)that has been manipulated, using the invention's compositions and/ormethods, at a different dosage and/or for a different duration and/orvia a different route of administration compared to the first subject.In one embodiment, the first and second samples (or subjects) may be thesame, such as where the effect of different regimens (e.g., of dosages,duration, route of administration, etc.) of the invention's compositionsand/or methods is sought to be determined on one sample (or subject). Inanother embodiment, the first and second samples (or subjects) may bedifferent, such as when comparing the effect of the invention'scompositions and/or methods on one sample (subject), for example apatient participating in a clinical trial and another individual in ahospital.

The terms “reduce,” “inhibit,” “diminish,” “suppress,” “decrease,” andgrammatical equivalents (including “lower,” “smaller,” etc.) when inreference to the level of any molecule (e.g., amino acid sequence, andnucleic acid sequence, antibody (such as IgG anti-F protein antibody,and anti-G protein antibody), etc.), cell (such as memory B cells),and/or phenomenon (e.g., the level of RSV neutralizing antibodies, thetiter RSV neutralizing antibodies, avidity, disease symptom, virusfunction, virion binding to receptors, virus uptake into cells,uncoating of the viral genomes in endosomes, infectivity by the virus,virus pathology (VEP), susceptibility to infection (VES) by the virus,transmission of the virus, etc.) in a first sample (or in a firstsubject) relative to a second sample (or relative to a second subject),mean that the quantity of molecule, cell and/or phenomenon in the firstsample (or in the first subject) is lower than in the second sample (orin the second subject) by any amount that is statistically significantusing any art-accepted statistical method of analysis. In oneembodiment, the quantity of molecule, cell and/or phenomenon in thefirst sample (or in the first subject) is at least 10% lower than, atleast 25% lower than, at least 50% lower than, at least 75% lower than,and/or at least 90% lower than the quantity of the same molecule, celland/or phenomenon in the second sample (or in the second subject). Inanother embodiment, the quantity of molecule, cell, and/or phenomenon inthe first sample (or in the first subject) is lower by any numericalpercentage from 5% to 100%, such as, but not limited to, from 10% to100%, from 20% to 100%, from 30% to 100%, from 40% to 100%, from 50% to100%, from 60% to 100%, from 70% to 100%, from 80% to 100%, and from 90%to 100% lower than the quantity of the same molecule, cell and/orphenomenon in the second sample (or in the second subject). In oneembodiment, the first sample (or the first subject) is exemplified by,but not limited to, a sample (or subject) that has been manipulatedusing the invention's compositions and/or methods. In a furtherembodiment, the second sample (or the second subject) is exemplified by,but not limited to, a sample (or subject) that has not been manipulatedusing the invention's compositions and/or methods. In an alternativeembodiment, the second sample (or the second subject) is exemplified by,but not limited to, a sample (or subject) that has been manipulated,using the invention's compositions and/or methods, at a different dosageand/or for a different duration and/or via a different route ofadministration compared to the first subject. In one embodiment, thefirst and second samples (or subjects) may be the same, such as wherethe effect of different regimens (e.g., of dosages, duration, route ofadministration, etc.) of the invention's compositions and/or methods issought to be determined on one sample (or subject). In anotherembodiment, the first and second samples (or subjects) may be different,such as when comparing the effect of the invention's compositions and/ormethods on one sample (subject), for example a patient participating ina clinical trial and another individual in a hospital.

“Substantially the same,” “without substantially altering,”“substantially unaltered,” and grammatical equivalents, when inreference to the level of any molecule (e.g., amino acid sequence, andnucleic acid sequence, antibody (such as IgG, anti-F protein antibody,and anti-G protein antibody), etc.), cell (such as memory B cells),and/or phenomenon (e.g., the level of RSV neutralizing antibodies, thetiter RSV neutralizing antibodies, avidity, disease symptom, virusfunction, virion binding to receptors, virus uptake into cells,uncoating of the viral genomes in endosomes, infectivity by the virus,virus pathology (VEP), susceptibility to infection (VES) by the virus,transmission of the virus, etc.) means that the quantity of molecule,cell, and/or phenomenon in the first sample (or in the first subject) isneither increased nor decreased by a statistically significant amountrelative to the second sample (or in a second subject). Thus in oneembodiment, the quantity of molecule, cell, and/or phenomenon in thefirst sample (or in the first subject) is from 90% to 100% (including,for example, from 91% to 100%, from 92% to 100%, from 93% to 100%, from94% to 100%, from 95% to 100%, from 96% to 100%, from 97% to 100%, from98% to 100%, and/or from 99% to 100%) of the quantity in the secondsample (or in the second subject).

Reference herein to any numerical range expressly includes eachnumerical value (including fractional numbers and whole numbers)encompassed by that range. To illustrate, and without limitation,reference herein to a range of “at least 50” includes whole numbers of50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, etc., and fractional numbers50.1, 50.2 50.3, 50.4, 50.5, 50.6, 50.7, 50.8, 50.9, etc. In a furtherillustration, reference herein to a range of “less than 50” includeswhole numbers 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, etc., andfractional numbers 49.9, 49.8, 49.7, 49.6, 49.5, 49.4, 49.3, 49.2, 49.1,49.0, etc. In yet another illustration, reference herein to a range offrom “5 to 10” includes each whole number of 5, 6, 7, 8, 9, and 10, andeach fractional number such as 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8,5.9, etc. In another example, the term “at least 95%” includes eachnumerical value (including fractional numbers and whole numbers) from95% to 100%, including, for example, 95%, 96%, 97%, 98%, 99% and 100%.

DESCRIPTION OF THE INVENTION

A major issue for development of RSV vaccines is that the vast majorityof the human population has experienced an RSV infection by 2 years ofage (37). In addition, most people experience RSV infections repeatedlyduring their lifetimes indicating that RSV infection does not stimulateeffective memory responses for neutralizing antibodies (37). However,any preexisting immunity could impact the effectiveness of a vaccine.Thus a successful vaccine candidate must stimulate high titers ofneutralizing antibody in the face of this preexisting immunity. Theinvention solves this problem by providing methods for using VLPvaccines containing a stabilized pre-fusion respiratory syncytial virus(RSV) F protein to stimulate RSV neutralizing antibodies in pre-immunesubjects.

Data herein demonstrates that RSV primed animals respond differently toRSV vaccines than naïve mice. This suggests that vaccine candidatestested in naïve animals may yield results not applicable to the vastmajority of the human population. This further suggests that vaccinecandidates need to be tested in previously infected animals or humans(which comprise the majority of the human population).

Data herein further demonstrates that RSV vaccine candidates containingthe pre-fusion F protein are far superior to vaccines containing thepost-fusion F protein, particularly in RSV primed animals suggestingthat successful vaccine candidates should contain a stabilizedpre-fusion F protein, as described herein.

The exemplary experiments described herein (Examples 1-12) assessed thegeneration of protective immune responses in mice previously infectedwith RSV by the invention's virus-like particle (VLP) vaccines thatcontain a stabilized pre-fusion form of the RSV F protein, in comparisonwith control stabilized post-fusion F protein. Data herein demonstratethat a single immunization of RSV-experienced animals with theinvention's stabilized pre-fusion F protein VLP stimulated high titersof neutralizing antibody while a single injection of a post-fusion Fprotein VLP or a second RSV infection only weakly stimulatedneutralizing antibody titers. These results show that prior RSVinfection induces neutralizing antibody memory responses, which can beactivated by pre-F protein VLPs but not by post-F protein VLPs or asubsequent infection. Thus the F protein conformation has a major impacton enhancing production of neutralizing antibodies in RSV-experiencedanimals. Furthermore, although both VLPs contained the same RSV Gprotein, the invention's pre-F VLP stimulated significantly highertiters of total anti-G protein IgG than the post-F VLP in both naïve andRSV-experienced animals. Thus the F protein conformation also influencesanti-G protein responses.

The invention is further described as follows under (A) Vaccines, (B)RSV Neutralizing Antibody Titers and Durability, (C) Anti-F ProteinAntibody And Anti-G Protein Antibody, (D) Spleen Memory B cells, and (E)Avidity.

(A) Vaccines

The invention provides a vaccine comprising recombinant chimericNewcastle Disease virus-like particles (ND VLPs) that contain a chimericprotein comprising, in operable combination, (1) stabilized pre-fusionRSV F protein ectodomain, (2) transmembrane (TM) domain of NDV Fprotein, and (3) cytoplasmic (CT) domain of NDV F protein (Example 1).In some embodiments, the vaccine further comprises, in operablecombination, one or more foldon sequence listed as SEQ ID NO:14. In analternate embodiment, the vaccine further comprises RSV G ectodomainsequence operably linked to the TM and CT domains of the NDV HN protein(Example 1).

(B) RSV Neutralizing Antibody Titers and Durability

In one embodiment, the invention provides a method for immunizing amammalian subject in need thereof (such as in need for immunizingagainst Respiratory Syncytial virus (RSV) infection), comprising, a)providing i) a first pre-immune mammalian subject containing RSVneutralizing antibodies, ii) a first composition comprising arecombinant chimeric ND VLP, exemplified by SEQ ID NO:02; FIG. 5B,(McGinnes Cullen et al. (2015) J Transl. Med 13:350) that contains achimeric protein comprising, in operable combination, 1) stabilizedpre-fusion RSV F protein ectodomain of McLellan et al. (19) (exemplifiedby SEQ ID NO:06; FIG. 7B), 2) transmembrane (TM) domain of NDV F protein(exemplified by SEQ ID NO:10; FIG. 9A), and 3) cytoplasmic (CT) domainof NDV F protein (exemplified by SEQ ID NO:12; FIG. 9B), and b)administering an immunologically effective amount of the firstcomposition to the first pre-immune subject to produce an immunizedsubject that comprises an increase in the level of the RSV neutralizingantibodies compared to the level of RSV neutralizing antibodies in thefirst pre-immune subject, thereby immunizing the first pre-immunesubject against the RSV infection (FIG. 1A, FIG. 12A, and FIG. 16A).

In one embodiment, the chimeric ND VLP further comprises, in operablecombination, a foldon sequence, exemplified by SEQ ID NO:14; FIG. 9C.

In one embodiment, the level of the RSV neutralizing antibodies in thefirst pre-immune subject does not prevent RSV infection of the firstpre-immune subject.

In a further embodiment, the level of the RSV neutralizing antibodies inthe immunized subject reduces (including 100% prevention) one or more ofRSV infection of the immunized subject compared to the first pre-immunesubject, and/or reduces (including 100% prevention) one or more symptomsof RSV infection, and/or reduces (including 100% prevention)susceptibility of the of the immunized subject to RSV infection comparedto the first pre-immune subject, and/or reduces (including 100%prevention) transmission of RSV infection from the immunized subject toa second subject, such as to a pre-immune subject or to anotherimmunized subject.

One surprising aspect of the invention's methods, is the increase in thelevel of the RSV neutralizing antibodies compared to the level of RSVneutralizing antibodies in the first pre-immune subject (FIG. 1A, andFIG. 12A).

In one particular embodiment, the increase in the level of the RSVneutralizing antibodies in the immunized subject is at least 100%,including at least from 100% to 10,000%, from 100% to 9,000%, from 100%to 8,000%, from 100% to 7,000%, from 100% to 6,000%, from 100% to5,000%, from 100% to 4,000%, from 100% to 3,000%, from 100% to 2,000%,and/or at least from 100% to 1,000% compared to the level of RSVneutralizing antibodies in the first pre-immune subject. An increase of“at least 100%” means at least a doubling. For example, at least a 100%increase in a level of 10 means at least a doubling to a level of atleast 20. Data herein in FIG. 1A show about 650% increase to a titer ofabout 4,500.

Another surprising aspect of the invention's methods, is that theimmunized subject comprises an increase in the level of the RSVneutralizing antibodies compared to the level of RSV neutralizingantibodies in a second (e.g., control) pre-immune subject that isinfected with RSV. In a particular embodiment, the increase in the levelof the RSV neutralizing antibodies in the immunized subject is at least100%, including at least from 100% to 10,000%, from 100% to 9,000%, from100% to 8,000%, from 100% to 7,000%, from 100% to 6,000%, from 100% to5,000%, from 100% to 4,000%, from 100% to 3,000%, from 100% to 2,000%,and/or at least from 100% to 1,000% compared to the level of RSVneutralizing antibodies in the second (e.g., control) pre-immune subjectthat is infected with RSV. An increase of “at least 100%” means at leasta doubling. For example, at least a 100% increase in a level of 10 meansat least a doubling to a level of at least 20. Data herein in FIG. 1Ashow about 650% increase to a titer of about 4,500.

In yet another surprising aspect of the invention's methods, theimmunized subject comprises an increase in the level of the RSVneutralizing antibodies compared to the level of RSV neutralizingantibodies in a second (e.g., control) pre-immune subject that istreated with a second composition comprising a control chimeric ND VLP(McGinnes Cullen et al. (2015) J Transl. Med 13:350) (SEQ ID NO:04, FIG.6B) that contains, in operable combination 1) stabilized post-fusion RSVF protein ectodomain of McLellan et al. (19) (exemplified by SEQ IDNO:08; FIG. 8B), 2) transmembrane domain of NDV F protein (exemplifiedby SEQ ID NO:10; FIG. 9A), and 3) cytoplasmic domain of NDV F protein(exemplified by SEQ ID NO:12; FIG. 9B), wherein the increase in thelevel of the RSV neutralizing antibodies in the immunized subject is atleast 100%, including at least from 100% to 10,000%, from 100% to9,000%, from 100% to 8,000%, from 100% to 7,000%, from 100% to 6,000%,from 100% to 5,000%, from 100% to 4,000%, from 100% to 3,000%, from 100%to 2,000%, and/or at least from 100% to 1,000% compared to the second(e.g., control) pre-immune subject that is treated with the secondcomposition. An increase of “at least 100%” means at least a doubling.For example, at least a 100% increase in a level of 10 means at least adoubling to a level of at least 20.

While not intending to limit the level of the neutralizing antibodytiter to a particular value, in one embodiment, the immunized subjectcomprises a titer of at least 700, including at least from 700 to10,000, from 800 to 10,000, from 900 to 10,000, from 1,000 to 10,000,from 2,000 to 10,000, from 3,000 to 10,000, from 4,000 to 10,000, from5,000 to 10,000, from 6,000 to 10,000, from 7,000 to 10,000, and/or atleast from 9,000 to 10,000. In one particular embodiment, the titer ofRSV neutralizing antibodies is about 4,500. FIG. 1A shows about 650%increase to a titer of about 4,500.

A further surprising aspect of the invention's methods is that the levelof the RSV neutralizing antibodies after a single administration of adose of the first composition to the first pre-immune subject issubstantially the same as the level of RSV neutralizing antibodies aftertwice administering the dose of the first composition to a control naïvesubject that has undetectable levels of RSV neutralizing antibodies(FIG. 1A, and FIG. 12A).

While not intending to limit the dosage of the compositions used in theinvention's methods, in one embodiment, the immunologically effectiveamount of the first composition comprises from 1 microgram to 50micrograms of the chimeric ND VLP (exemplified by SEQ ID NO:02; FIG.5B), including from 1 to 49, from 1 to 48, from 1 to 47, from 1 to 46,from 1 to 45, from 1 to 44, from 1 to 43, from 1 to 42, from 1 to 41,from 1 to 40, from 1 to 39, from 1 to 38, from 1 to 37, from 1 to 36,from 1 to 35, from 1 to 34, from 1 to 33, from 1 to 32, from 1 to 31,from 1 to 20, from 1 to 19, from 1 to 18, from 1 to 17, from 1 to 16,from 1 to 15, from 1 to 14, from 1 to 13, from 1 to 12, from 1 to 11,from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, andfrom 1 to 5 micrograms of the chimeric ND VLP. In a particularembodiment, the immunologically effective amount of the firstcomposition comprises from 4 micrograms to 45 micrograms of the chimericND VLP. In a particular embodiment, the immunologically effective amountof the first composition comprises 30 micrograms of the chimeric ND VLP.Data herein show administration of 30 micrograms ND VLP containingapproximately 7 micrograms of pre-fusion RSV F protein ectodomain(Example 2, FIG. 1A).

In a further embodiment, the immunologically effective amount of thefirst composition comprises from 0.1 microgram to 20 micrograms of thechimeric protein (exemplified by SEQ ID NO:06; FIG. 7B), including from0.5 to 20, from 1 to 20, from 1 to 19, from 1 to 18, from 1 to 17, from1 to 16, from 1 to 15, from 1 to 14, from 1 to 13, from 1 to 12, from 1to 11, and from 1 to 10 of the chimeric protein. In a particularembodiment, the immunologically effective amount of the firstcomposition comprises seven (7) micrograms of the chimeric protein. Dataherein show administration of 30 micrograms ND VLP containingapproximately 7 micrograms of pre-fusion RSV F protein ectodomain(Example 2, FIG. 1A).

In some embodiments, the immunologically effective composition may beadministered more than once to further boost immunity against RSV.

In one embodiment, the invention's methods comprise determining thelevel of the RSV neutralizing antibodies in any one or more of thesubjects described herein (e.g., naïve subject, and/or pre-immunesubject, and/or subject treated with one or more of the invention'sPre-F/F VLPs and/or subject treated with Post-F/F VLPs, and/or subjectinfected with RSV). In particular embodiments, the inventions' methodscomprise comparing the levels of the RSV neutralizing antibodies in anytwo or more of the subjects described herein (e.g., naïve subject,and/or pre-immune subject, and/or subject treated with one or more ofthe invention's Pre-F/F VLPs and/or subject treated with Post-F/F VLPs,and/or subject infected with RSV).

In some embodiments, the method further comprises comparing the level ofthe RSV neutralizing antibodies in the immunized subject to the level ofthe RSV neutralizing antibodies in one or more test subjects selectedfrom a) the first pre-immune subject, b) a second (e.g., control)pre-immune subject that is infected with RSV, c) a second (e.g.,control) pre-immune subject that is treated with a control secondcomposition comprising a chimeric ND VLP (exemplified by SEQ ID NO:04;FIG. 6B; McGinnes Cullen et al. (2015) J Transl. Med 13:350] thatcontains, in operable combination 1) stabilized post-fusion RSV Fprotein ectodomain (19) (exemplified by SEQ ID NO:08; FIG. 8B), 2)transmembrane domain of NDV F protein (exemplified by SEQ ID NO:10; FIG.9A), and 3) cytoplasmic domain of NDV F protein (exemplified by SEQ IDNO:12; FIG. 9B), wherein detecting an increase in the level of the RSVneutralizing antibodies in the immunized subject compared to the levelof the RSV neutralizing antibodies in the one or more test subjectsindicates that the immunized subject is immunized against the RSVinfection.

In a particular embodiment, the method further comprises detecting inthe immunized subject a reduction in one or more of (a) level of RSVinfection, (b) one or more symptoms of RSV infection, (c) susceptibilityto RSV infection, and (d) transmission of RSV infection, compared to thefirst pre-immune subject.

In a further embodiment, the invention's methods are carried out underconditions wherein the level of the RSV neutralizing antibodies after asingle administration of the invention's Pre-F/F VLPs to the firstpre-immune subject is higher than the level of RSV neutralizingantibodies after a single administration of the invention's ND VLPs to anaïve subject, and/or to a pre-immune subject infected with RSV, and/orto a pre-immune subject treated with Post-F/F VLPs. Data herein inExample 10, FIG. 16A-B, demonstrates that a single injection of VLPsinto RSV primed mice stimulated much higher NA titers than in naïvemice. Additionally, data herein in Example 10, FIG. 16A-B, demonstratesthat in RSV primed animals, (1) A single injection of the invention'sPre-F/F VLPs resulted in 7 and 3.7 fold higher neutralizing antibodytiters than post-F/F VLPs (days 128 vs 220, respectively), (2) theinvention's Pre-F/F VLPs immunization resulted in 12 and 8 fold highertiters (day 128 vs 220 respectively) than a second RSV Infection and (3)Post-F/F VLPs resulted in 1.8 fold to 2.3 fold higher titers (day 128 vsDay 220) than a second RSV infection. In naïve mice, two injections ofthe invention's Pre-F/F VLPs resulted in titers approximately 50% thatof a single immunization in RSV primed animals.

Data herein in Example 10, FIG. 16A-B also shows that immunization ofanimals previously infected with RSV (to mimic the vast majority of thehuman population) with the invention's Pre-F/F VLPs is far superior toimmunization with post-F/F VLPs. The absolute levels of neutralizingantibodies stimulated by the invention's Pre-F/F VLPs at day 128 were 7fold higher than levels stimulated by Post-F/F VLPs.

With respect to antibody durability, immunization of animals previouslyinfected with RSV with the invention's Pre-F/F VLPs is far superior toimmunization with post-F/F VLPs in terms of absolute levels ofneutralizing antibodies at later times. Thus, in one embodiment, theinvention's methods are carried out under conditions wherein the levelof the RSV neutralizing antibodies after a single administration of theinvention's Pre-F/F VLPs to the first pre-immune subject is higher forat least a period of from about 30 to about 220 days after the singleadministration than the level of RSV neutralizing antibodies after asingle administration of a second Post-F/F VLPs to a second pre-immunesubject. Data herein in Example 10, FIGS. 16A-B-18A-B, demonstrates thatRSV NA titers are quite stable with time after a single injection of theinvention's pre-F/F VLPs. Data herein in Example 10 also shows thatbecause titers after Pre-F/F VLP immunization were 7 fold and 12 foldhigher than the titers after Post-F/F VLP or RSV immunization at day128, the titers after the pre-F/F VLP immunization at day 220 were stillmuch higher than titers after Post-F/F VLP or RSV immunization (3.7 foldand 8 fold).

(C) Anti-F Protein Antibody And Anti-G Protein Antibody

In one embodiment, the invention's methods comprise determining thelevel of anti-F protein antibody (such as anti-F protein IgG) and/or ofanti-G protein antibody (such as anti-G protein IgG) in any one or moreof the subjects described herein (e.g., naïve subject, and/or pre-immunesubject, and/or subject treated with one or more of the invention'sPre-F/F VLPs and/or subject treated with Post-F/F VLPs, and/or subjectinfected with RSV). In particular embodiments, the inventions' methodscomprise comparing the levels of anti-F protein antibody (such as anti-Fprotein IgG) and/or of anti-G protein antibody (such as anti-G proteinIgG) in any two or more of the subjects described herein (e.g., naïvesubject, and/or pre-immune subject, and/or subject treated with one ormore of the invention's Pre-F/F VLPs and/or subject treated withPost-F/F VLPs, and/or subject infected with RSV).

In a further embodiment, the invention's methods are carried out underconditions wherein the levels of pre-F specific IgG in a pre-immunesubject treated with the invention's Pre-F/F VLPs or Post-F/F VLPs isthe same but the levels are lower than the levels of post-F specific IgGin a pre-immunized subject treated with Pre-F/F VLPs or Post-F/F VLPs(FIGS. 13A and 13B).

Importantly, FIG. 13A-B shows that even though the IgG titers (specificfor pre-F or post-F) are the same after Pre-F/F VLP or Post-F/F VLPimmunization of pre-immune animals, nonetheless, the neutralizingantibody titers are quite different (FIGS. 1A-B, 12A-B, 16A-B). Thismeans that the specificities of the antibodies stimulated by the two VLPimmunzations are quite different. That is, the Pre-F/F VLPs robustlystimulate neutralizing antibodies , whereas the Post-F/F VLPs do not.This key observation probably explains why all previous vaccinecandidates have failed since, until recently, all candidates containedprimarily post F protein which stimulated IgG titers but these IgGs werepoorly neutralizing.

Data herein in Example 10, FIG. 17A-D shows that a single immunizationof RSV primed animals with VLPs resulted in 10 fold higher IgG titerscompared to two immunizations with VLPs in naïve animals. Example 10,FIG. 17A-D also shows that a single immunization of RSV primed animalswith VLPs resulted in 10 fold higher IgG titers than RSV infections.This shows that total anti-F IgG levels also remained stable with time,and that VLPs stimulated very durable total anti-F IgG antibodies inboth RSV primed and naïve animals.

In particular embodiments, the invention's methods comprise using NDVLPs that include, in operable combination, RSV G protein (e.g., RSV Gectodomain). This is exemplified by VLP-H/G+Pre-F/F.

In some embodiments, the invention's methods include measuring the levelof antibody that is specific for the RSV G protein (e.g., RSV Gectodomain) in any one or more of the subjects described herein (e.g.,naïve subject pre-immune subject, and/or subject treated with one ormore of the invention's Pre-F/F VLPs and/or subject treated withPost-F/F VLPs, and/or subject infected with RSV). In particularembodiments, the inventions' methods comprise comparing the levels ofantibody that is specific for the RSV G protein (e.g., RSV G ectodomain)in any two or more of the subjects described herein (e.g., naïvesubject, and/or pre-immune subject, and/or subject treated with one ormore of the invention's Pre-F/F VLPs and/or subject treated withPost-F/F VLPs, and/or subject infected with RSV).

Thus, in one embodiment, the invention's methods are carried out underconditions wherein the level of antibody that is specific for the RSV Gprotein (e.g., RSV G ectodomain) in a pre-immune subject after a singleadministration of the invention's Pre-F/F VLPs is higher than the levelof antibody that is specific for the RSV G protein (e.g., RSV Gectodomain) in the pre-immune subject prior to the administration step,and/or after a single administration of ND Post-F/F VLPs, and/or in apre-immune subject infected with RSV, and/or in a naïve subject. Forexample, data in Example 7, FIG. 14A-B shows that in RSV-experiencedanimals, a single VLP immunization with either the VLP-H/G+Pre-F/F orthe VLP-H/G+Post-F/F considerably increased the anti-G protein antibodytiters and this increase was approximately four fold over thatstimulated by a prime and boost with either VLP in naïve animals. Also,data in Example 10, FIG. 18A-C, shows that with respect to antibodylevels in RSV primed animals: (1) VLPs stimulated 7 fold (theinvention's pre-F/F VLPs) and 5 fold (post-F/F VLPs) higher anti-Gprotein titers than in VLPs in naïve animals, (2) VLPs stimulated 65fold (pre-F VLPs) and 25 fold (post-F/F VLPs) higher anti-G proteinantibody titers than two consecutive RSV infections, and (3) Pre-F VLPsstimulated 2.6 fold higher anti-G protein antibody titers than post-FVLPs. Importantly, these differences in anti-G antibody levels show thatthe presence of the pre-F protein in the invention's Pre-F/F VLP has asignificant influence on levels of immune responses to the G protein,which is a protein that a role in protective responses to RSV.

In a particular embodiment the inventions methods are carried out underconditions wherein a single administration of the invention's ND VLPthat comprises RSV G protein (e.g., RSV G ectodomain) to a firstpre-immune mammalian subject increases the level of anti-G proteinantibody compared to the level of anti-G protein antibody in a secondpre-immune subject that is immunized with a second compositioncomprising chimeric ND Post-F/F VLPs. Data herein in Example 7, FIG.14A-B, shows the surprising result that the levels of anti-G proteinantibodies after a single VLP immunization of RSV-experienced animals orafter a VLP prime and boost of naïve animals were significantlydifferent depending upon the VLP used although both VLPs containedsimilar amounts of the same H/G protein (FIG. 10A-D, panels FIG. 10A andFIG. 10D). VLPs containing the pre-fusion F protein simulatedsignificantly higher titers of anti-G protein antibody than the VLPscontaining the post-F protein.

In one embodiment, the invention's methods comprise determining the RSVtiter in lungs after RSV challenge in any one or more of the subjectsdescribed herein (e.g., naïve subject, and/or pre-immune subject, and/orsubject treated with one or more of the invention's Pre-F/F VLPs and/orsubject treated with Post-F/F VLPs, and/or subject infected with RSV).In particular embodiments, the inventions' methods comprise comparingthe RSV titer in lungs in any two or more of the subjects describedherein (e.g., naïve subject, and/or pre-immune subject, and/or subjecttreated with one or more of the invention's Pre-F/F VLPs and/or subjecttreated with Post-F/F VLPs, and/or subject infected with RSV).

In a particular embodiment, the methods of the invention are carried outunder conditions wherein the pre-immune subject that is immunized withthe invention's Pre-F/F VLPs has a lower RSV titer in lungs than a naïvesubject to which the invention's Pre-F/F VLP have not been administered.Data herein in Example 8, FIG. 15, show that while RSV titers wereobtained in the lungs of unprimed, unimmunized controls (lane A), novirus was detected at the limits of detection in lungs of immunizedanimals. These results demonstrate that immunization of primed animalswith the invention's Pre-F/F VLP protected them from RSV replication.

(D) Spleen Memory B Cells

In one embodiment, the invention's methods comprise determining thelevel of splenic memory B cells in any one or more of the subjectsdescribed herein (e.g., naïve subject, and/or pre-immune subject, and/orsubject treated with one or more of the invention's Pre-F/F VLPs and/orsubject treated with Post-F/F VLPs, and/or subject infected with RSV).In particular embodiments, the inventions' methods comprise comparingthe levels of level of splenic memory B cells in any two or more of thesubjects described herein (e.g., naïve subject, and/or pre-immunesubject, and/or subject treated with one or more of the invention'sPre-F/F VLPs and/or subject treated with Post-F/F VLPs, and/or subjectinfected with RSV).

Thus, in one embodiment, the invention's methods are carried out underconditions wherein the level of splenic memory B cells in a pre-immunesubject that is immunized with the invention's Pre-F/F VLPs is higherthan the level of splenic memory B cells in a pre-immune subject that isimmunized with Post-F/F VLPs. Data herein in Example 11, FIG. 19A-Bdemonstrates that pre-F/F protein in VLPs stimulated higher levels ofsplenic memory B cells than post-F/F VLPs in RSV experienced animals.

In another embodiment, the invention's methods are carried out underconditions wherein the level of splenic memory B cells in a pre-immunesubject that is immunized with the invention's Pre-F/F VLPs is higherthan the level of splenic memory B cells after RSV infection of apre-immune subject and/or after RSV infection of a naïve subject. Dataherein in Example 11, FIG. 20A-B, shows that the invention's Pre-F/FVLPs stimulated higher levels of splenic memory B cells than RSV in RSVexperienced animals as well as naïve mice.

(E) Avidity

In one embodiment, the invention's methods comprise determining thelevel of avidity of the RSV anti-F or G IgG antibodies in any one ormore of the subjects described herein (e.g., naïve subject, and/orpre-immune subject, and/or subject treated with one or more of theinvention's Pre-F/F VLPs and/or subject treated with Post-F/F VLPs,and/or subject infected with RSV). In particular embodiments, theinventions' methods comprise comparing the levels of avidity of the RSVantibodies in any two or more of the subjects described herein (e.g.,naïve subject, and/or pre-immune subject, and/or subject treated withone or more of the invention's Pre-F/F VLPs and/or subject treated withPost-F/F VLPs, and/or subject infected with RSV).

In one embodiment, the invention's methods are carried out underconditions wherein the avidity of the RSV antibodies in a pre-immunesubject immunized with the invention's Pre-F/F VLPs is higher thanavidity of RSV antibodies in a pre-immune subject infected with RSV.Data herein in Example 12, FIGS. 22A-B show that avidity of the pre-Fspecific antibodies stimulated by VLPs is higher than the avidity ofantibodies stimulated by two RSV infections, i.e., VLP Stimulatedantibodies are 2 fold more resistant to 3 M urea than RSV stimulatedantibodies. Also, FIGS. 22A and B show that in RSV primed animals, theinvention's Pre-F/F VLPs stimulated higher avidity antibodies thanPost-F/F VLPs, i.e., the invention's Pre-F/F VLP stimulated antibodiesare 1.7 fold more resistant to 7 M urea than Post-F/F VLP stimulatedantibodies.”

In a particular embodiment, the invention's methods are carried outunder conditions wherein the avidity of the anti-G protein antibody inthe pre-immunized subject that is treated with the invention's Pre-F/FVLPs is higher than the avidity of anti-G protein antibody in apre-immune mammalian subject infected with RSV. Data herein in Example12, FIG. 21C shows that the invention's pre-F/F VLPs stimulated higheravidity anti-G protein antibodies than RSV infections.

EXPERIMENTAL

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof.

EXAMPLE 1 Materials and Methods A. Cells, Virus, Plasmids

ELL-0 (avian fibroblasts) (CLR-12203), Vero cells (CLR-1586), COS-7cells (CLR-1651), and Hep2 cells (CCL-23) were obtained from theAmerican Type Culture Collection. Expi293F cells were obtained fromThermoFisher/Invitrogen (A14527). ELL-0 cells, Vero cells, COS-7 cells,and Hep2 cells were grown in DMEM (Invitrogen 1195-073) supplementedwith penicillin, streptomycin (Invitrogen 15140-122), and 5% (Verocells) or 10% fetal calf serum (Invitrogen 10437-028). Expi293F cellswere grown in Expi293 media (ThermoFisher/Gibco/Invitrogen A1435101).RSV, A2 strain, was obtained from Dr. Robert Finberg.

VLPs containing the RSV F and G proteins were formed with the Newcastledisease virus (NDV) core proteins NP and M^(18,33) and contain the NDVHN/RSV G Amino Acid Sequence (SEQ ID NO:16) (FIG. 24B) encoded by theNDV HN/RSV G DNA Sequence (SEQ ID NO:15) (FIG. 24A). The cDNAs encodingthe NDV NP and M protein have been previously described³⁴. The RSV F andG proteins are incorporated into these VLPs by constructing chimeraprotein genes composed of ectodomains of the G or F glycoproteins fusedto the transmembrane (TM) and cytoplasmic (CT) domains of the NDV HNprotein or NDV F glycoprotein, respectively. These NDV domainsspecifically interact with the NDV NP and M protein resulting inefficient incorporation of the chimera proteins into VLPs.

The construction, expression, and incorporation of the chimera proteinNDVHN/RSVG (H/G) into VLPs have been previously described¹⁹. Theconstruction, expression, and incorporation into VLPs of the stabilizedpre-fusion F protein (Pre-F/F DS-Cav1) to generate VLP-H/G+Pre-F/F, andthe stabilized post-fusion F protein (Post-F/F) to createVLP-H/G+Post-F/F have been previously described²¹.

The construction of genes encoding the soluble pre-F protein, thesoluble post-F protein, and the soluble G protein used for target inELISA was previously described²¹.

B. Polyacrylamide Gel Electrophoresis, Silver Staining, and WesternAnalysis

Proteins were resolved on 8% Bis-Tris gels (NuPage,ThermoFisher/Invitrogen WB1001/WG1002)). Silver staining of proteins inthe polyacrylamide gels was accomplished as recommended by themanufacturer (ThermoFisher/Pierce 24600). Quantification of NP, M,different forms of F/F, H/G protein, and soluble pre-F, post-F, andsoluble G was accomplished after their separation in polyacrylamide gelsfollowed by silver staining or by Western blots of the proteins as wellas protein standards as previously described^(35,36). For Westernanalysis, proteins in the polyacrylamide gels were transferred to PVDFmembranes using dry transfer (iblot, ThermoFisher/Invitrogen iB401001).Proteins were detected in the blots using anti-RSV HR2 peptide antibodyor anti-RSV antibody.

C. Antibodies

RSV F monoclonal antibody clone 131-2A (Millipore MAB8599) was used inRSV plaque assays. Monoclonal antibody (mAb) 1112, mAb 1200, mAb 1243,were generous gifts of Dr. J. Beeler ³⁷and used to verify F proteinconformations, and mAb D25 and mAb motavizumab, generous gifts of Dr. J.McLellan,¹⁴, were used for ELISA analysis of VLPs and soluble Fproteins. Anti-RSV F protein HR2 antibody used for Western Blots is apolyclonal antibody specific to the HR2 domain of the RSV F protein¹⁸.Anti-RSV G protein antibody is a polyclonal antibody raised against apeptide containing G protein amino acids 180-198 (ThermoFisherPA5-22827). Secondary antibodies against goat (A5420), mouse (A5906) andrabbit IgG (A0545) were purchased from Sigma.

D. VLP Preparation, Purification, and Characterization

For preparations of VLPs to be used as immunogens (VLP-H/G+Pre-F/F,VLP-H/G+Post-F/F), ELL-0 cells growing in T-150 flasks were transfectedwith cDNAs encoding the NDV M protein, NP, the chimeric proteins H/G,and either Pre-F/F or Post-F/F as previously described^(18,19). At 24hours post-transfection, heparin (Sigma, H4784) was added to the cellsat a final concentration of 10 μg/ml¹⁹ to inhibit rebinding of releasedVLPs to cells. At 72, 96, and 120 hours post-transfection, cellsupernatants were collected and VLPs purified by sequential pelletingand sucrose gradient fractionation as previously described^(18,19,35).Concentrations of proteins in the purified VLPs were determined bysilver-stained polyacrylamide gels and by Western analysis using markerproteins for standard curves^(18,35). The conformation of F protein inthe VLP preparations was verified by reactivity to mAbs.

E. Preparation of Soluble F Proteins

Expi293F cells were transfected with pCAGGS vector containing sequencesencoding the soluble pre-F protein or the soluble post-F protein. Atfive to six days post transfection, total cell supernatants werecollected and cell debris removed by centrifugation. Pre-fusion andpost-fusion polypeptides were then purified on columns using the His tagand then the strep tag as previously described¹⁵.

F. Quantification of Soluble F Protein and VLP Associated F Protein

Determinations of amounts of RSV F protein in VLPs or in soluble Fprotein preparations were accomplished by Western blots using anti-HR2antibody for detection and comparing the signals obtained with astandard curve of purified F proteins as previously described³⁵.Quantification of amounts of soluble G protein was determined on Westernblots using anti-RSV G protein antibody for detection.

G. Preparation of RSV, RSV Plaque Assays, and Antibody Neutralization

RSV was grown in Hep2 cells^(18,19), and RSV plaque assays wereaccomplished on Vero cells as previously described²¹. Antibodyneutralization assays in a plaque reduction assay have been previouslydescribed^(21,22). Neutralization titer was defined as the reciprocal ofthe dilution of serum that reduced virus titer by 50%.

H. Animals, Animal Immunization, and RSV Challenge

Mice, 4-week-old female BALB/c, from Taconic laboratories (BALB-F), werehoused (groups of 5) under pathogen-free conditions in microisolatorcages at the University of Massachusetts Medical Center animal quarters.Female mice were used in order to assess the potential of VLPs formaternal immunization. Protocols requiring open cages were accomplishedin biosafety cabinets. BALB/c mice were immunized by intramuscular (IM)inoculation of 30 μg total VLP protein (5 microgram (μg) F protein) in0.05 ml of THE (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA)containing 10% sucrose. For infections with RSV, the animals werelightly anesthetized with isoflurane and then infected by intranasal(IN) inoculation of 50 microliter (μ1) of RSV (1×10⁷ pfu/ml). All animalprocedures and infections were performed in accordance with theUniversity of Massachusetts Medical School IACUC and IBC approvedprotocols.

I. ELISA Protocols

For determination of anti-F protein or anti-G protein serum antibodytiters, blood was obtained from immunized animals by tail vein nicks andcentrifuged in BD microtainer serum separator tubes (ThermoFisher365967) to remove blood cells. For ELISA, wells of microtiter plates(ThermoFisher/Costar 2797) were coated with either purified solublepre-fusion F protein, soluble post-fusion F protein, or soluble Gprotein and incubated for 24 hours at 4° C. Wells were then incubated inPBS-2% BSA for 16 hours. Different dilutions of sera, in 0.05% Tween and2% BSA, were added to each well and incubated for 2 hours at roomtemperature. After six washes in PBS, sheep anti-mouse antibody coupledto HRP (Sigma A5906) was added in 50 μl PBS-2% BSA and incubated for 1.5hours at room temperature. Bound HRP was detected by adding 50 μl TMB(3,3′5,5′-tetramethylbenzidin, TheimoFisher34028) and incubating for5-20 minutes at room temperature until blue color developed. Thereaction was stopped with 50 μl 2N sulfuric acid. Color was read inSpectraMax Plus Plate Reader (Molecular Devices) using SoftMax Prosoftware. Amounts of IgG bound to the wells was calculated using astandard curve generated using defined amounts of purified IgG³⁸.

J. Statistical Analysis

Statistical analyses (student T test) of data were accomplished usingGraph Pad Prism 6 software.

EXAMPLE 2 Neutralizing RSV Antibody is Stimulated by ND VLPs ContainingStabilized RSV Pre-F/F Protein

The materials and methods for cells, viruses, plasmids, preparation ofsoluble forms of the pre-fusion and post-fusion F proteins and Gprotein, antibodies, polyacrylamide e gel electrophoresis, silverstaining, western analysis, VLP preparation, VLP purification, VLPcharacterization, ELISA protocols, preparation of RSV, RSV plaqueassays, antibody neutralization, animals, animal immunization, RSVchallenge, lung and nose viral titration, pulmonary histopathology, andstatistical analysis, are previously described (36).

A schematic of testing VLP immunogenicity as a Vaccine in an animal(exemplified by mouse) is shown in FIG. 4. Briefly, mice were immunizedwith ND VLPs containing stabilized RSV pre-F/F protein operably fused tothe foldon sequence (SEQ ID NO:14, FIG. 9C). The stabilized pre-fusionRSV F protein ectodomain was previously described by McLellan et al.(19) and shown in FIG. 3. The RSV pre-F/F chimera protein contains RSVpre-F protein ectodomain operably linked to both the NDV F proteintransmembrane (TM) domain and the NDV F protein cytoplasmic (CT) domain(FIG. 3).

ND VLPs containing stabilized RSV pre-F/F protein operably fused to thefoldon sequence (SEQ ID NO:02, FIG. 5B) were constructed as previouslydescribed (34, 36) (FIG. 3).

FIGS. 1A-B and 12A-B show that VLPs with the appropriate conformation ofF protein can stimulate high titers of neutralizing antibodies in thepresence of preexisting immunity: FIG. 1 A and FIG. 12A, shows that, inRSV pre-immune animals, pre-F/F VLPs stimulated neutralizing antibodiestiters significantly higher than VLPs containing the post-F/F protein ora second RSV infection. Previously reported results (34) of immunizationof naïve animals, after a prime and a prime-boost with pre-F/F VLPs,post-F/F VLPs, or RSV is shown in FIG. 1B and FIG. 12B, for comparison.

In the experiment with pre-immune animals (FIG. 1A-B and FIG. 12A-B,left panel FIG. 12A), groups of 5 mice were infected with RSV byintranasal inoculation (FIG. 4). Ninety-five days later, one group wasimmunized with Pre-F/F VLPs, another group immunized with Post-F/F VLPs,and a third group given a second infection with RSV. The results showthe following:

First, in RSV pre-immune animals (left panel A), pre-F/F VLPs stimulatedsignificantly higher neutralizing Ab titers than post-F/F VLPs or RSV.Importantly, that is, pre-F/F VLPs can stimulate high titers ofneutralizing antibodies in the presence of pre-existing immunity to RSV.

Second, post-F/F VLPs stimulated slightly higher titers than a secondRSV infection.

Third, the titers after a single pre-F/F VLP immunization of pre-immunemice were comparable to titers in sera of naïve mice after a prime and aboost with pre-F/F VLPs (titers of 4500-right panel B) (34), suggestingthe pre-F/F VLPs stimulated memory responses in the RSV-pre-immuneanimal. However, the post-F/F VLP immunization of pre-immune micestimulated titers more comparable to titers after single post-F/Fimmunization of naïve mice (titers of 600 vs 150) rather than titersobtained after a prime and boost immunization with post-F/F VLPs (titersof 2700, FIG. 1A-B, right panel FIG. 1B).

In conclusion, these results show that the pre-F/F VLPs should be a veryeffective vaccine for RSV pre-immune individuals, who make up most ofthe human population.

EXAMPLE 3 Characterization of Protein Content of VLP Stocks

VLPs, based on Newcastle disease virus (NDV) core proteins andcontaining the RSV G protein (e.g., RSV G ectodomain) and either thepre-fusion or post-fusion forms of the RSV F protein, were generated bytransfection of ELL-0 cells with plasmids encoding NDV M protein, NDVNP, the H/G chimera protein¹⁹, and either the Pre-F/F or the Post-F/Fchimera proteins to generate stocks of VLP-H/G+Pre-F/F orVLP-H/G+Post-F/F²¹. The protein content of the two purified VLPpreparations was quantified by Western blots and antibody binding to thepurified VLPs. FIG. 10A-D, panel FIG. 10A, shows a Western blot ofproteins in the two VLP preparations probed with anti-RSV F (lanes 1 and2) or anti-RSV G antibodies (lane 3 and 4). The results show that stocksof the two VLPs had equivalent levels of Pre-F/F and Post-F/F chimeraproteins and equivalent levels of the H/G chimera protein. The two Fprotein chimeras are different sizes since the Pre-F/F contains theinserted foldon sequence and the Post-F/F chimera has a deletion of nineamino acids. The H/G chimera protein resolves into heterogeneous speciesdue to inefficient glycosylation of the RSV G protein sequences aspreviously described^(19,21). To further verify protein concentrationsin VLPs, a monoclonal antibody that will bind either form of the RSV Fprotein, motavizumab^(13,23) binds equally to the two VLPs (FIG. 10A-D,panel FIG. 10B) verifying that the two VLPs have assembled equivalentlevels of F protein. However, a monoclonal antibody specific for site 4)present only in the pre-fusion form of F protein but not in the postfusion form¹⁴ binds only VLP-H/G+Pre-F/F and not VLP-H/G+Post-F/F (FIG.10A-D, panel FIG. 10C), a result verifying the conformation of the pre-Fprotein and the post-F protein in the two VLPs. A polyclonal antibodyraised against a G protein derived peptide bound equivalently to twodifferent concentrations of the two VLPs (FIG. 10A-D, panel FIG. 10D)verifying that the two VLPs have the same amount of H/G chimera protein.

EXAMPLE 4 Infection and Immunization

To assess the generation of neutralizing antibody responses in micepreviously infected with RSV, three groups of five mice were prepared byinfection with RSV by intranasal inoculation. After ninety-five days,one group was immunized with VLP-H/G+Pre-F/F, another group immunizedwith VLP-H/G+Post-F/F, and a third group was infected a second time withRSV (FIG. 11A). To directly compare responses in previously infectedmice with those in naïve mice, in parallel, groups of five naïve micewere immunized in a prime (day 0) and a boost (day 100) with theVLP-H/G+Pre-F/F, with the VLP-H/G+Post-F/F, or RSV infection (FIG. 11B).Serum samples were obtained from each mouse at different times startingat day 0.

EXAMPLE 5 Neutralization Titers in Previously Infected and Naïve Animals

To determine the effect of previous RSV infection on generation ofneutralizing antibodies (NA), the neutralization titers in pooled seraof mice at different times after an RSV prime and VLP immunization weredetermined using an in vitro plaque reduction assay (FIG. 12A-B, panelFIG. 12A). A single injection of these RSV-experienced animals withVLP-H/G+Pre-F/Fs stimulated significantly higher NA titers thanVLP-H/G+Post-F/Fs or a second RSV infection. VLP-H/G+Pre-F/Fimmunization resulted in titers of approximately 4000 by day 128 whileVLP-H/G+Post-F/Fs stimulated NA titers of approximately 600 at day 128,only slightly higher than a second RSV infection.

FIG. 12A-B, panel FIG. 12B, illustrates, in parallel groups of naïvemice, the neutralization titers in animals after a prime and after aboost with the either VLP-H/G+Pre-F/Fs, VLP-H/G+Post-F/Fs, or after oneor two RSV infections. These results are very similar to resultspreviously reported for VLP immunization of naïve animals²¹. In a primeimmunization, the VLP-H/G+Pre-F/Fs stimulated significantly highertiters than the VLP-H/G+Post-F/Fs or a single RSV infection. A boostwith VLP-H/G+Pre-F/Fs increased titers to approximately 4000 while aVLP-H/G+Post-F/Fs boost resulted in titers of approximately 2500. Twoconsecutive RSV infections produced NA titers of approximately 200.

EXAMPLE 6 Total Anti-F IgG Titers after Immunization of RSV-ExperiencedAnimals

To determine if the differences in the NA titers after a singleimmunization of RSV-experienced mice with VLP-H/G+Pre-F/Fs orVLP-H/G+Post-F/Fs could be accounted for by differences in total anti-Fprotein antibody, the amounts of total anti-F protein IgG in the sera ofthe two groups were determined at each time point and compared to IgGlevels in RSV infected mice. The titers of anti-F protein IgG that bindto the soluble pre-fusion F protein are shown in FIG. 13A-D, panels FIG.13A, while the binding of serum IgG to the soluble post-fusion F proteinis shown in panel FIG. 13B. The results show that a single immunizationwith VLP-H/G+Pre-F/Fs or VLP-H/G+Post-F/Fs stimulated virtuallyequivalent titers of IgG specific for soluble pre-fusion F protein orsoluble post-fusion F protein. A second RSV infection did stimulateanti-F protein IgG but the levels were 10 fold lower than thosestimulated by both VLPs. Thus different levels of total anti-F proteinIgG cannot account for the differences in NA titers after immunizationwith the VLP-H/G+Pre-F/Fs or VLP-H/G+Post-F/Fs.

The IgG levels specific for pre-F and post-F targets generated in naïvemice after a prime VLP immunization and after a boost immunization areshown in FIG. 13A-D, panels FIG. 13C and D, respectively. Levels of IgGspecific to the pre-F target are lower than those specific to the post-Ftarget after immunization with either VLP. Interestingly, the levels ofIgG specific to both pre-F and post-F targets after a prime and boostare approximately ten fold lower than levels generated after RSV primingand a single VLP immunization

EXAMPLE 7 Total Anti-G Protein IgG Titers After Immunization ofRSV-Experienced or Naïve Animals

Antibodies specific for the RSV G protein also have a role in protectiveresponses to RSV infection²⁴⁻²⁷. Thus it was of interest to determinethe influence of previous RSV infection on generation of anti-G proteinantibodies. The titers of anti-G protein IgG antibodies in the parallelsets of naïve and RSV-experienced mice were determined using soluble Gprotein as target in ELISA. FIG. 14A-B, panel FIG. 14A, shows antibodytiters in sera after VLP-H/G+Pre-F/F or VLP-H/G+Post-F/F immunization ofRSV-experienced mice while panel B shows titers after a prime and aboost of naïve mice with VLP-H/G+Pre-F/Fs or VLP-H/G+Post-F/Fs or RSV.In both naïve and RSV-experienced mice, anti-G protein antibody levelswere extremely low after a single RSV infection or after a single VLPimmunization. A second RSV infection in both sets of mice only minimallystimulated anti-G protein antibody levels. In contrast, VLP prime andboost immunization of naïve mice substantially increased anti-G proteinantibody titers. Importantly, in RSV-experienced animals, a single VLPimmunization with either the VLP-H/G+Pre-F/F or the VLP-H/G+Post-F/Fconsiderably increased the anti-G protein antibody titers and thisincrease was approximately four fold over that stimulated by a prime andboost with either VLP in naïve animals.

A surprising result was that the levels of anti-G protein antibodiesafter a single VLP immunization of RSV-experienced animals or after aVLP prime and boost of naïve animals were significantly differentdepending upon the VLP used although both VLPs contained similar amountsof the same H/G protein (FIG. 10A-D, panels FIG. 10A and D)²¹. VLPscontaining the pre-fusion F protein simulated significantly highertiters of anti-G protein antibody than the VLPs containing the post-Fprotein.

EXAMPLE 8 Protection from RSV Challenge

To determine if a single VLP immunization of RSV-experienced animalscould protect them from RSV replication in lungs after RSV challenge,mice were challenged with RSV 125 days after VLP immunization. FIG. 15shows titers of virus in lung homogenates. While good titers wereobtained in the unprimed, unimmunized controls (lane A), no virus wasdetected at the limits of detection in lungs of immunized animals. Theresults demonstrated that immunization with either VLP of RSV primedanimals protected them from RSV replication.

Results of the challenge of naïve, immunized mice have been previouslypublished²¹.

EXAMPLE 9 Discussion Of Results In Examples 3-15

The goal of the experiments in Examples 1-15 was to mimic humanpopulations by assessing immune responses to the instant invention's VLPvaccines in mice previously infected with RSV.

When comparing NA titers, the data showed that in animals previouslyinfected with RSV, a single immunization with VLP-H/G+Pre-F/Fsstimulated significantly higher NA titers than a single immunizationwith VLP-H/G+Post-F/Fs or a second RSV infection. The NA titers after asingle VLP-H/G+Pre-F/F immunization of previously infected mice werecomparable to titers in sera of naïve mice only after both a prime and aboost with VLP-H/G+Pre-F/Fs. This result suggests that RSV infectiondoes induce potent neutralizing antibody memory responses that can beactivated by the VLP-H/G+Pre-F/F immunization but not byVLP-H/G+Post-F/Fs or a second RSV infection.

A recent paper from Gilman, et al²⁹ supports the idea that RSV infectioninduces pre-F memory cells, and shows that RSV infection does indeedinduce significant levels of memory B cells that encode high titerneutralizing antibodies, at least in humans. Data herein show that RSVinfection can induce protective memory in mice but a subsequentinfection cannot activate these memory B cells.

In contrast to results with VLP-H/G+Pre-F/Fs, a single immunization withVLP-H/G+Post-F/F in RSV infected mice resulted in NA titers more similarto those observed after a single VLP-H/G+Post-F/F immunization of naïvemice. However, a prime/boost immunization of naïve mice withVLP-H/G+Post-F/Fs stimulated good NA titers, titers that wereapproximately 50% that simulated by VLP-H/G+Pre-F/Fs. That theVLP-H/G+Post-F/F immunization in RSV-experienced mice did not stimulatethese higher NA titers suggests that the RSV infection may not inducememory responses to some determinants present in the VLP-H/G+Post-F/Fspreventing high NA titers with a single VLP-H/G+Post-F/F immunization.

Thought it was possible that differences in NA titers afterVLP-H/G+Pre-F/F or VLP-H/G+Post-F/F immunization of RSV-experienced micewere due to differences in total levels of anti-F protein antibodies insera of the animals, data herein, however, clearly demonstrated that thelevels of total anti-F protein antibodies in animals immunized withVLP-H/G+Pre-F/Fs were virtually identical to levels of total antibodiesin VLP-H/G+Post-F/F immunized animals. Thus the differences in NA titersin the VLP-H/G+Pre-F/F and VLP-H/G+Post-F/F immunized, RSV-experiencedanimals must be due to qualitative differences between the populationsof anti-F protein antibodies in the two groups of animals. Immunizationof RSV-experienced mice with either VLP did result in much higher anti-Fprotein antibody titers than a second RSV infection indicating that asecond RSV infection very poorly activates secondary antibody responsesin contrast to the VLP immunization. It is noteworthy that naïve micehave total IgG anti-F protein levels after a prime and boost with VLPssimilar to that observed after RSV infection. However, total antibodylevels by day 128 in these naïve mice were approximately ten fold lowerthan total levels in the VLP immunized RSV-experienced mice.

Studies of protective immune responses to RSV have largely focused onthe role of the F protein. However, antibodies to G protein do have arole in protection from RSV induced disease. The G protein centralregion contains a conserved sequence that is a mimic of the chemokineCX3C (fractalkine).²⁷ The G protein competes for the binding of CX3C toits receptor, CX3CR1, inhibiting immune responses to RSV in a number ofways that enhance the pathology of the infection^(27,31 26). Antibodiesto the G protein CX3C sequence block G protein binding to the CX3CR1moderating RSV disease. Importantly, antibody to this CX3C sequencedecrease enhanced respiratory disease that results from RSV challenge ofFI-RSV vaccinated animals³². Treatment with mAb to CX3C sequencedecreased symptoms in RSV infected mice²⁵. For these reasons, levels oftotal anti-G protein IgG in naïve mice immunized with VLPs or infectedwith RSV were compared to levels of these antibodies in RSV-experiencedmice after VLPs immunization or a second RSV infection. Data herein showthat in naïve mice a single RSV infection or one VLP immunization(prime) both generate anti-G protein antibodies very poorly. However, asingle immunization with either VLP in RSV-experienced animals resultedin significant titers of anti-G protein antibodies suggesting that RSVinfection does induce memory responses to the G protein. In contrast, asecond infection with RSV results in barely detectable levels of anti-Gprotein antibody suggesting that RSV cannot effectively stimulate thisanti-G protein memory. One surprising result of this analysis is thatthe VLP-H/G+Pre-F/F induced significantly higher titers of anti-Gprotein IgG than the VLP-H/G+Post-F/F in both naïve and RSV-experiencedanimals. It is important to point out that the two different VLPs,VLP-H/G+Pre-F/Fs and VLP-H/G+Post-F/Fs, contain the same H/G chimeraprotein and in the same amounts²¹. These results suggest that theconformation of the F protein in VLPs influences induction andstimulation of total anti-G IgG.

In summary, results of assessing levels of anti-F or anti-G proteinantibodies in RSV-experienced animals vs naïve animals suggests that RSVinfection can induce memory responses but infection is defective instimulating or activating that memory. Further, these results indicatethat the conformation of the F protein in a vaccine candidate hassignificant impact on the nature of anti-RSV immune responses in micepreviously infected with RSV.

EXAMPLE 10 Levels and Durability of Neutralizing Antibody Titers

To address the question of how does prior infection with RSV affectimmunization with the invention's VLPs, the protocol of FIG. 11A-B wasused to examine neutralizing antibody (NA) levels, as well as thedurability of NA titers in sera. The potential influence of theconformation of the RSV F protein on these responses was also examined.Data is shown in FIGS. 16A-B-18A-C.

As previously demonstrated (FIGS. 1A-B and 12A-B) FIG. 16A-Bdemonstrates that pre-F/F VLPs stimulate higher titers of neutralizingantibodies and more durable levels of neutralizing antibodies in RSVexperienced animals than Post-F/F VLPs. In particular, in RSV primedanimals, (1) A single injection of Pre-F/F VLPs resulted in 7 and 3.7fold higher neutralizing antibody titers than post-F/F VLPs (days 128 vs220, respectively), (2) Pre-F/F VLPs immunization resulted in 12 and 8fold higher titers (day 128 vs 220, respectively) than a second RSVInfection and (3) Post-F/F VLPs resulted in 1.8 fold to 2.3 fold highertiters (day 128 vs Day 220) than a second RSV infection. In naïve mice,two injections of Pre-F/F VLPs resulted in titers approximately 50% thatof a single immunization in RSV primed animals. Thus, this data showsthat immunization of animals previously infected with RSV (to mimic thevast majority of the human population) with Pre-F/F VLPs is far superiorto immunization with post-F/F VLPs. The absolute levels of neutralizingantibodies stimulated by Pre-F/F VLPs at day 128 are 7 fold higher thanlevels stimulated by Post-F/F VLPs.

FIG. 16A-B also shows that neutralizing antibody titers stimulated byPre-F/F VLPs, post-F/F VLPs, and RSV are similarly durable. Titers afterPre-F/F VLP Immunization dropped 1.8 fold while those after Post-F/F VLPimmunization dropped 1.5 fold. Titers after RSV infection dropped 1.2fold. Because titers after Pre-F/F VLP immunization were 7 fold and 12fold higher than the titers after Post-F/F VLP or RSV immunization atday 128, the titers after the pre-F/F VLP immunization at day 220 arestill much higher than titers after Post-F/F VLP or RSV immunization(3.7 fold and 8 fold). This data shows that immunization of animalspreviously infected with RSV with Pre-F/F VLPs is far superior toimmunization with post-F/F VLPs in terms of absolute Levels ofneutralizing antibodies at later times.

FIG. 17A-D shows that total anti-F IgG titers are unaffected by Fconformation. In particular, FIG. 17A-D shows that a single immunizationof RSV primed animals with VLPs resulted in 10 fold higher IgG titerscompared to two immunizations with VLPs in naïve animals. A singleimmunization of RSV primed animals with VLPs resulted in 10 fold higherIgG titers than RSV infections. Thus, total anti-F IgG levels remainedstable with time in all animals. This demonstrates that VLPs stimulatevery durable total anti-F IgG antibodies in both RSV primed and naïveanimals

As previously shown in FIG. 14A-B, FIG. 18A-C shows that VLPs stimulatehigher anti-G protein titers in RSV experienced animals than in naïveanimals, and that the invention's pre-F VLPs stimulate higher anti-Gprotein titers than post-F VLPs. In particular, FIG. 18A-C shows that,with respect to antibody levels in RSV primed animals: (1) VLPsstimulated 7 fold (pre-F/F VLPs) and 5 fold (post-F/F VLPs) higheranti-G protein titers than in VLPs in naïve animals, (2) VLPs stimulated65 fold (pre-F VLPs) and 25 fold (post-F/F VLPs) higher anti-G proteinantibody titers than two consecutive RSV infections, and (3) Pre-F VLPsstimulated 2.6 fold higher anti-G protein antibody titers than post-FVLPs.

FIG. 18A-C also show that, with respect to antibody levels in naïveanimals: (1) Two Pre-F/F VLP immunizations stimulated 10 fold higheranti-G protein antibody titers than two consecutive RSV infections, (2)Two immunizations with post-F/F VLPs stimulated 5 fold higher anti-Gprotein antibody titers than RSV infection, and (3) Two immunizationswith Pre-F/F VLPs consistently stimulated nearly 2 fold higher titersthan two immunizations with post-F/F VLPs.

Importantly, the differences in anti-G protein antibody levels shown inFIG. 18A-C were obtained using pre-F/F and Post-F/F VLPs that containedthe same G protein in the same amounts. The results suggest that thepresence of the F protein, and particularly the pre-F protein in VLPs,has a significant influence on levels of immune responses to the Gprotein. Since it has been shown by other investigators that G proteinhas a role in protective responses to RSV, this finding has asignificant impact on vaccine formulation.

Regarding the durability of anti-G protein protein antibodies, FIG.18A-C shows that, in RSV primed animals, anti-G protein antibodiesstimulated by Pre-F/F VLPs were less durable than antibodies stimulatedby post-F/F VLPs (decreases with time of 2.6 and 1.25 fold,respectively). FIG. 18A-C also shows that, in naïve animals, titers ofanti-G protein antibodies stimulated by pre-F/F VLPs were less durablethan titers of anti-G protein antibodies stimulated by Post-F/F VLPs.Pre-F/F VLP titers decreased with time four fold while titers stimulatedwith Post-F/F VLPs decreased by two fold.

In sum, the data demonstrates that: (1) with respect to the levels ofNA, a single injection of VLPs into RSV primed mice stimulates muchhigher NA titers than in naïve mice, (2) with respect to the durabilityof NA titers in sera, NA titers are quite stable with time after asingle injection of VLPs, particularly the invention's pre-F/F VLPs, (3)F protein conformation influences the levels of NA antibodies sincepre-F/F protein in VLPs significantly increased NA titers in micepreviously infected with RSV compared to post-F/F VLPs, and (4) Fprotein conformation influences anti-G protein IgG responses, sincepre-F/F protein in VLPs increased anti-G protein IgG responses in RSVexperienced animals compared to post-F/F VLPs or RSV infection.

EXAMPLE 11 Anti-Pre-F Protein Antibody Secreting Splenic B Cells

RSV infections, in contrast to the vast majority of virus infections,are defective in memory responses so one can get RSV many times duringlife. The effect of immunization with VLPs containing pre-F protein orpost-F protein on memory B cells secreting antibody to pre-F protein andpost-F protein, respectively, was determined. Memory B cells wereactivated by infecting animals 4 days prior to sacrifice for titrationof memory B cells. Data is shown in FIGS. 19A-B and 20A-B.

FIG. 19A-B shows that with respect to the pre-F protein target, thetiter of memory B cells secreting pre-F antibodies is significantlyhigher after a single VLP Immunization of RSV primed mice compared totwo injections of VLPs in naïve mice. Immunization with Pre-F/F VLPsresulted in higher memory B cell titers compared to post-F/F VLPs.

FIG. 20A-B shows that similarly to the pre-F protein target, withrespect to the post-F protein target, the titer of memory B cellssecreting post-F antibodies is significantly higher after a single VLPimmunization of RSV primed animals compared to two injections of VLPs innaïve mice. Immunization with Pre-F/F VLPs resulted in higher memory Bcell titers compared to post-F/F VLPs in RSV primed animals. FIG. 20A-Balso shows that A single injection of VLPs stimulate higher levels ofsplenic memory B cells in RSV primed mice than two injections in naïveanimals, and that VLPs stimulated higher levels of splenic memory Bcells than RSV in RSV experienced animals as well as naïve mice.

This data also demonstrates that F protein conformation influencessplenic memory B cells, since pre-fusion F/F protein in VLPs stimulatedhigher levels of splenic memory B cells than post-F/F VLPs in RSVexperienced animals.

Thus, the invention's VLPs stimulate memory responses which are superiorto those stimulated by RSV infections and are, thus superior inproviding long term protection from RSV infection.

EXAMPLE 12 Avidity/Stability of Antigen-Antibody Complexes

We determined the avidity/stability of antigen-antibody complexes inincreasing urea for pre-F protein target, post-F protein target, and Gprotein target. Data is shown in FIG. 21A-C, FIG. 22A-D, and FIG. 23A-B.

Importantly, FIG. 21A-C shows VLPs, particularly pre-F VLPs, stimulatedhigher avidity anti-F antibodies than RSV infection in RSV primed mice.Indeed, in RSV primed animals, a single dose of VLPs stimulated muchhigher avidity anti-F antibodies than two consecutive RSV infections.

FIG. 21A-C also shows that the avidity of anti-F specific antibodiesstimulated by Pre-F/F VLPs is significantly higher than the avidity ofanti-F antibodies stimulated by post-F/F VLPs, and that the avidity ofanti-G protein antibodies stimulated by either VLP or RSV infection tosoluble G protein is quite weak.

FIGS. 22A-B show that avidity of both pre-F and post-F specificantibodies stimulated by VLPs is higher than antibodies stimulated bytwo RSV infections, i.e., VLP Stimulated antibodies are 2 fold and 1.4fold more resistant to 3 M urea than RSV stimulated antibodies.

FIGS. 22, A and B, show that in RSV primed animals, Pre-F/F VLPsstimulated higher avidity antibodies than Post-F/F VLPs, i.e., Pre-F/FVLP stimulated antibodies are 1.7 fold more resistant to 7 M urea thanPost-F/F VLP stimulated antibodies.

FIG. 23A shows that avidity of anti-G protein antibodies stimulated byVLPs or RSV infection was low, i.e., anti-G protein antibody-G proteincomplexes are very sensitive to urea.

FIGS. 21A-C-23A-B also demonstrate that F protein conformationinfluences the avidity/stability of antigen-antibody complexes, sinceVLPs, particularly pre-F/F VLPs, stimulate higher avidity anti-Fantibodies than RSV infection.

References Cited in “Background of the Invention,” “Description of theInvention,” and “Example 2”

-   1. Karron R A. 2008. Respiratory syncytial virus and parainfluenza    virus vaccines. In A P S, A O W, P A O (ed.), Vaccines, 5th ed.    Saunders-Elsevier.-   2. Falsey A R, Hennessey P A, Formica M A, Cox C, Walsh E E. 2005.    Respiratory syncytial virus infection in elderly and high-risk    adults. N. Engl. J. Med 352:1749-1759.-   3. Falsey A R, Walsh E E. 2000. Respiratory syncytial virus    infection in adults. Clin Microbiol Rev 13:371-384.-   4. Han L L, Alexander J P, Anderson L J. 1999. Respiratory syncytial    virus pneumonia among the elderly: an assessment of disease burden.    J Infect Dis 179:25-30.-   5. Raboni S M, Nogueira M B, Tsuchiya L R, Takahashi G A, Pereira L    A, Pasquini R. 2003. Respiratory tract viral infections in bone    marrow transplant patients. Transplant. 76:142-146.-   6. Hall C B, Long C E, Schnabel K D. 2001. Respiratory syncytial    virus infections in previously healthy working adults. Clin Infect    Dis 33:792-796.-   7. Collins P L, Graham B S. 2007. Viral and host factors in human    respiratory syncytial virus pathogenesis. J. Virol. 82:2040-2055.-   8. Littel-van den Hurk S D, Mapletoft J W, Arsic N,    Kovacs-Nolan J. 2007. Immunopathology of RSV infection: prospects    for developing vaccines without this complication. Rev Med. Virol.    17:5-34.-   9. Openshaw P J, Culley F J, Olszewska W. 2002. Immunopathogenesis    of vaccine-enhanced RSV disease. Vaccine. 20:27-31.-   10. Openshaw P J, Tregoning J S. 2005. Immune responses and disease    enhancement during respiratory syncytial virus infection. Clin    Microbiol Rev 18:541-555.-   11. Jardetsky T S, Lamb R A. 2004. A class act. Nature. 427.-   12. Perrone L A, Ahmad A, Veguilla V, Lu X, Smith G, Katz J M,    Pushko P, Tumpey T M. 2009. Intranasal Vaccination with 1918    Influenza Virus-Like Particles Protects Mice and Ferrets from Lethal    1918 and H5N1 Influenza Virus Challenge. J. Virol. 83:5726-5734.-   13. Lamb R A, Parks G D. 2007. Paramyxoviridae: The Viruses and    Their Replication, p. 1450-1496. In Knipe D M, Howley P M, Griffin D    E, Lamb R A, Martin M A, Roizman B, Strauss S E (ed.), Fields    Virology, Fifth Edition ed, vol. 1. LippincottWilliams &Wilkins,    Philadelphia.-   14. Arav-Boger R, Willoughby R E, Pass R F, Zong J C, Jang W J,    Alcendor D, Hayward G S. 2002. Polymorphisms of the cytomegalovirus    (CMV)-encoded tumor necrosis factor-alpha and beta-chemokine    receptors in congenital CMV disease. J Infect Dis 186:1057-1064.-   15. Blair K S, Smith B W, Mitchell D G, Morton J, Vythilingam M,    Pessoa L, Fridberg D, Zametkin A, Sturman D, Nelson E E, Drevets W    C, Pine D S, Martin A, Blair R J. 2007. Modulation of emotion by    cognition and cognition by emotion. Neuroimage 35:430-440.-   16. Swanson K A, Settembre E C, Shaw C A, Dey A K, Rappuoli R, Mandl    C W, Dormitzer P R, Carfi A. 2011. Structural basis for immunization    with postfusion respiratory syncytial virus fusion F glycoprotein    (RSV F) to elicit high neutralizing antibody titers. Proc. Natl.    Acad. Sci USA. 108:9619-9624.-   17. McLellan J S, Yang Y, Graham B S, Kwong P D. 2011. Structure of    Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion    Conformation Reveals Preservation of Neutralizing Epitopes. J.    Virol. 85:7788-7796.-   18. McLellan J S, Chen M, Leung S, Graepel K W, Du X, Yang Y, Zhou    T, Baxa U, Yasuda E, Beaumont T, Kumar A, Modjarrad K, Zheng Z, Zhao    M, Xia N, Kwong P D, Graham B S. 2013. Structure of RSV Fusion    Glycoprotein Trimer Bound to a Prefusion-Specific Neutralizing    Antibody. Science 340:1113-1117.-   19. McLellan J S, Chen M, Joyce M G, Sastry M, Stewart-Jones G B E,    Yang Y, Zhang B, Chen L, Srivatsan S, Zheng A, Zhou T, Graepel K W,    Kumar A, Moin S, Boyington J C, Chuang G-Y, Soto C, Baxa U, Bakker A    Q, Spits H, Beaumont T, Zheng Z, Xia N, Ko S-Y, Todd J-P, Rao S,    Graham B S, Kwong P D. 2013. Structure-Based Design of a Fusion    Glycoprotein Vaccine for Respiratory Syncytial Virus. Science    342:592-598.-   20. Smith G, Raghunandan R, Wu Y, Liu Y, Massare M, Nathan M, Zhou    B, Lu H, Boddapati S, Li J, Flyer D, Glenn G. 2012. Respiratory    Syncytial Virus Fusion Glycoprotein Expressed in Insect Cells Form    Protein Nanoparticles That Induce Protective Immunity in Cotton    Rats. PLoS ONE 7:e50852.-   21. Ngwuta J O, Chen M, Modjarrad K, Joyce M G, Kanekiyo M, Kumar A,    Yassine H M, Moin S M, Killikelly A M, Chuang G-Y, Druz A, Georgiev    I S, Rundlet E J, Sastry M, Stewart-Jones G B E, Yang Y, Zhang B,    Nason M C, Capella C, Peeples M E, Ledgerwood J E, McLellan J S,    Kwong P D, Graham B S. 2015. Prefusion F-specific antibodies    determine the magnitude of RSV neutralizing activity in human sera.    Science Transl. Med. 7:309ra162.-   22. Magro M, Mas V, Chappell K, Vazquez M, Cano O, Luque D, Tenon M    C, Melero J A, Palomo C. 2012. Neutralizing antibodies against the    preactive form of respiratory syncytial virus fusion protein offer    unique possibilities for clinical intervention. Proc. Natl. Acad.    Sci. 109:3089-3094.-   23. Glezen W, Taber L H, Frank A L, Kasel J A. 1986. Risk of primary    infection and reinfection with respiratory syncytial virus.    American J. of Dis. of Children 140:543-546.-   24. Hall C B. 2001. Respiratory syncytial virus and parainfluenza    virus. N Engl J Med 344:1917-1928.-   25. Power U F. 2008. Respiratory syncytial virus (RSV) vaccines—Two    steps back for one leap forward. J Clin Virol 41:38-44.-   26. Pulendran B, Ahmed R. 2011. Immunological mechanisms of    vaccination. Nat Immunol 12:509-517.-   27. Murawski M R, McGinnes L W, Finberg R W, Kurt-Jones E A, Massare    M, Smith G, Heaton P M, Fraire A, Morrison T G. 2010. Newcastle    disease virus-like particles containing respiratory syncytial virus    G protein induced protection in BALB/c mice with no evidence of    immunopathology. J. Virol. 84:1110-1123.-   28. McGinnes L W, Gravel K A, Finberg R W, Kurt-Jones E A, Massare M    J, Smith G, Schmidt M R, Morrison T G. 2011. Assembly and    immunological properties of Newcastle disease virus-like particles    containing the respiratory syncytial virus F and G proteins. J.    Virol. 85:366-377.-   29. Bachmann M F, Jennings G T. 2010. Vaccine delivery: a matter of    size, geometry, kinetics and molecular patterns. Nat Rev Immunol    10:787-796.-   30. McGinnes Cullen L, Schmidt M R, Kenward S A, Woodland R T,    Morrison T G. 2015. Murine Immune Responses to Virus-Like    Particle-Associated Pre- and Postfusion Forms of the Respiratory    Syncytial Virus F Protein. J. of Virol. 89:6835-6847.

31. Schmidt M R, McGinnes L W, Kenward S A, Willems K N, Woodland R T,Morrison T G. 2012. Long term and memory immune responses in miceagainst Newcastle disease virus-like particles containing respiratorysyncytial virus glycoprotein ectodomains. J. Virol. 86:11654-11662.

-   32. Morrison T G. 2010. Newcastle disease virus-like particles as a    platform for the development of vaccines for human and agricultural    pathogens. Future Virol. 5:545-554.-   33. Schmidt M R, McGinnes-Cullen L W, Kenward S A, Willems K N,    Woodland R T, Morrison T G. 2014. Modification of the respiratory    syncytial virus F protein in virus-like particles impacts generation    of B cell memory. J Virol 88:10165-10176.-   34. McGinnes-Cullen L, Schmidt M R, Kenward S A, Woodland R T,    Morrison T G. 2015. Murine Immune Responses to Virus-Like    Particle-Associated Pre- and Postfusion Forms of the Respiratory    Syncytial Virus F Protein. J of Virol. 89:6835-6847.-   35. McLellan J S, Chen M, Kim A, Yang Y, Graham B S, Kwong    P D. 2010. Structural basis of respiratory syncytial virus    neutralization by motavizumab. Nat Struct Mol Biol 17.-   36. Cullen L M, Blanco J C G, Morrison T G. 2015. Cotton rat immune    responses to virus-like particles containing the pre-fusion form of    respiratory syncytial virus fusion protein. J. Transl. Med.

13:1-13.

-   37. Hall C B, Simoes E A F, Anderson L J. 2013. Clinical and    Epidemiologic Features of Respiratory Syncytial Virus, p. 39-58. In    Anderson L T, Graham B S (ed.), Challenges and Opportunities for    Respiratory Syncytial Virus Vaccines, vol. 372. Springer,    Heidelberg, New York, Dordrecht, Londaon.-   38. McLellan J S, Chen M, Kim A, Yang Y, Graham B S, Kwong    P D. 2011. Structural basis of respiratory syncytial virus    neutralization by motavizumab. Nat Struct Mol Biol 17:248-250.

References Cited in Example 1, and Examples 3-9

-   1. Karron R A. Respiratory syncytial virus and parainfluenza virus    vaccines. In: Plotkin S A, Orenstein W A, Offit P, eds. Vaccines.    5th ed: Saunders-Elsevier; 2008:1146.-   2. Falsey A R, Hennessey P A, Formica M A, Cox C, Walsh E E.    Respiratory syncytial virus infection in elderly and high-risk    adults. N Engl J Med 2005; 352:1749-59.-   3. Falsey A R, Walsh E E. Respiratory syncytial virus infection in    adults. Clin Microbiol Rev 2000; 13:371-84.-   4. Han L L, Alexander J P, Anderson L J. Respiratory syncytial virus    pneumonia among the elderly: an assessment of disease burden. J    Infect Dis 1999; 179:25-30.-   5. Raboni S M, Nogueira M B, Tsuchiya L R, Takahashi G A, Pereira L    A, Pasquini R. Respiratory tract viral infections in bone marrow    transplant patients. Transplant 2003; 76:142-6.-   6. Hall C B, Long C E, Schnabel K D. Respiratory syncytial virus    infections in previously healthy working adults. Clin Infect Dis    2001; 33:792-6.-   7. Power U F. Respiratory syncytial virus (RSV) vaccines--Two steps    back for one leap forward. J Clin Virol 2008; 41:38-44.-   8. Graham B S. Biological challenges and technological opportunities    for respiratory syncytial virus vaccine development. Immunol Rev    2012; 239:149-66.-   9. Morrison T G, Walsh E E. Subunit and Virus-like Particle Vaccine    Approached for Respiratory Syncytial Virus. In: Anderson L J, Graham    B S, eds. Challenges and opportunities for respiratory syncytial    virus vaccines. Heidelberg, Berlin: Springer; 2013.-   10. Jardetsky T S, Lamb R A. A class act. Nature 2004; 427.-   11. Lamb R A, Parks G D. Paramyxoviridae: The Viruses and Their    Replication. In: Knipe D M, Howley P M, Griffin D E, et al., eds.    Fields Virology. Fifth Edition ed. Philadelphia: LippincottWilliams    &Wilkins; 2007:1450-96.-   12. Swanson K A, Settembre E C, Shaw C A, et al. Structural basis    for immunization with postfusion respiratory syncytial virus fusion    F glycoprotein (RSV F) to elicit high neutralizing antibody titers.    Proc Natl Acad Sci USA 2011; 108:9619-24.-   13. McLellan J S, Yang Y, Graham B S, Kwong P D. Structure of    Respiratory Syncytial Virus Fusion Glycoprotein in the Postfusion    Conformation Reveals Preservation of Neutralizing Epitopes. J Virol    2011; 85:7788-96.-   14. McLellan J S, Chen M, Leung S, et al. Structure of RSV Fusion    Glycoprotein Trimer Bound to a Prelusion-Specific Neutralizing    Antibody. Science 2013; 340:1113-7.-   15. McLellan J S, Chen M, Joyce M G, Sastry M, Stewart-Jones G B E,    Yang Y. Structure-based design of a fusion glycoprotein vaccine for    respiratory syncytial virus. Science 2013; 342:592-8.-   16. Hall C B, Simoes E A F, Anderson L J. Clinical and Epidemiologic    Features of Respiratory Syncytial Virus. In: Anderson L J, Graham B    S, eds. Challenges and Opportunities for Respiratory Syncytial Virus    Vaccines. Heidelberg, New York, Dordrecht, Londaon: Springer;    2013:39-58.-   17. Glezen W, Taber L H, Frank A L, Kasel J A. RIsk of primary    infection and reinfection with respiratory syncytial virus. American    J of Dis of Children 1986; 140:543-6.-   18. McGinnes L W, Gravel K A, Finberg R W, Kurt-Jones E A, Massare M    J, Smith G. Assembly and immunological properties of Newcastle    disease virus-like particles containing the respiratory syncytial    virus F and G proteins. J Virol 2011; 85:366-77.-   19. Murawski M R, McGinnes L W, Finberg R W, Kurt-Jones E A, Massare    M, Smith G. Newcastle disease virus-like particles containing    respiratory syncytial virus G protein induced protection in BALB/c    mice with no evidence of immunopathology. J Virol 2010; 84:1110-23.-   20. Bachmann M F, Jennings G T. Vaccine delivery: a matter of size,    geometry, kinetics and molecular patterns. Nat Rev Immunol 2010;    10:787-96.-   21. McGinnes-Cullen L, Schmidt M R, Kenward S A, Woodland R T,    Morrison T G. Murine Immune Responses to Virus-Like    Particle-Associated Pre- and Postfusion Forms of the Respiratory    Syncytial Virus F Protein. J of Virol 2015; 89:6835-47.-   22. Cullen L M, Blanco J C G, Morrison T G. Cotton rat immune    responses to virus-like particles containing the pre-fusion form of    respiratory syncytial virus fusion protein. J Transl Med 2015;    13:1-13.-   23. McLellan J S, Chen M, Kim A, Yang Y, Graham B S, Kwong P D.    Structural basis of respiratory syncytial virus neutralization by    motavizumab. Nat Struct Mol Biol 2011; 17:248-50.-   24. Boyoglu-Barnum S, Todd S O, Chirkova T, Barnum T R, Gaston K A,    Haynes L M. An anti-G protein monoclonal antibody treats RSV disease    more effectively than an anti-F monoclonal antibody in BALB/c mice.    Virology 2015; 483.-   25. Boyoglu-Barnum S, Todd S O, Chirkova T, et al. An anti-G protein    monoclonal antibody treats RSV disease more effectively than an    anti-F monoclonal antibody in BALB/c mice. Virology 2015;    483:117-25.-   26. Tripp R A. Pathogenesis of respiratory syncytial virus    infection. Viral Immunol 2004; 17:165-81.-   27. Tripp R A, Jones L P, Haynes L M, Zheng H, Murphy P M, Anderson    L J. CX3C chemokine mimicry by respiratory syncytial virus G    glycoprotein. Nat Immunol 2001; 2:732-8.-   28. Hall C B. Respiratory syncytial virus and parainfluenza virus. N    Engl J Med 2001; 344:1917-28.-   29. Gilman M S A, Castellanos C A, Chen M, et al. Rapid profiling of    RSV antibody repertoires from the memory B cells of naturally    infected adult donors. Sci Immunol 2016; 1:1879.-   30. Collins P L, Crowe J E. Respiratory syncytial virus and    metapneumovirus. 5 ed. Philadelphia: LippincottWilliams and Wilkins;    2007.-   31. Chirkova T, Boyoglu-Barnum S, Gaston K A, et al. Respiratory    Syncytial Virus G Protein CX3C Motif Impairs Human Airway Epithelial    and Immune Cell Responses. J of Virol 2013; 87:13466-79.-   32. Rey G U, Miao C, Caidi H, et al. Decrease in    Formalin-Inactivated Respiratory Syncytial Virus (FI-RSV) Enhanced    Disease with RSV G Glycoprotein Peptide Immunization in BALB/c Mice.    PLoS ONE 2013; 8:e83075.-   33. McGinnes L W, Pantua H, Laliberte J P, Gravel K A, Jain S,    Morrison T G. Assembly and biological and immunological properties    of Newcastle disease virus-like particles. J Virol 2010; 84:4513-23.-   34. McGinnes L W, Reitter J, Pantua H D, Morrison T G. Newcastle    disease virus: propagation, quantification, and storage: John Wiley    and sons, Inc; 2006.-   35. McGinnes L W, Morrison T G. Newcastle Disease Virus-Like    Particles: Preparation, Purification, Quantification, and    Incorporation of Foreign Glycoproteins. Current Protocols in

Microbiology: John Wiley & Sons, Inc.; 2013.

-   36. Gravel K A, McGinnes L W, Reitter J, Morrison T G. The    transmembrane domain sequence affects the structure and function of    the Newcastle disease virus fusion protein. J Virol 2011;    85:3486-97.-   37. Beeler J A, van Wyke Coelingh K. Neutralization epitopes of the    F glycoprotein of respiratory syncytial virus: effect of mutation    upon fusion function. J Virol 1989; 63:2941-50.-   38. McGinnes L W, Morrison T G. Current Protocols in Microbiology.    USA: Wiley; 2013.

Each and every publication and patent mentioned in the abovespecification is herein incorporated by reference in its entirety forall purposes. Various modifications and variations of the describedmethods and system of the invention will be apparent to those skilled inthe art without departing from the scope and spirit of the invention.Although the invention has been described in connection with specificembodiments, the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in the art and in fields related thereto are intended tobe within the scope of the following claims.

1. A method for immunizing a mammalian subject, comprising, a) providing i) a first RSV experienced mammalian subject containing Respiratory Syncytial Virus (RSV) neutralizing antibodies, wherein the first RSV experienced mammalian subject's experience is selected from the group consisting of being previously in contact with RSV. being previously infected by RSV, and being both previously in contact with RSV and previously infected by RSV, ii) a first composition comprising recombinant chimeric Newcastle Disease virus-like particles (ND VLPs) that contain a chimeric protein comprising, in operable combination, 1) stabilized pre-fusion RSV F protein ectodomain, 2) transmembrane (TM) domain of NDV F protein, and 3) cytoplasmic (CT) domain of NDV F protein, and b) administering an immunologically effective amount of the first composition to the first RSV experienced mammalian subject to produce a first immunized mammalian subject, wherein said administering is under conditions that increase the level of the RSV neutralizing antibodies in said first immunized mammalian subject.
 2. The method of claim 1, wherein the level of the RSV neutralizing antibodies in the first RSV experienced subject does not prevent RSV infection of the first RSV experienced subject.
 3. The method of claim 2, wherein the level of the RSV neutralizing antibodies in the first immunized subject reduces RSV infection of the first immunized subject compared to the first RSV experienced subject.
 4. The method of claim 2, wherein the level of the RSV neutralizing antibodies in the first immunized subject reduces one or more symptoms of RSV infection.
 5. The method of claim 2, wherein the level of the RSV neutralizing antibodies in the first immunized subject reduces susceptibility of the first immunized subject to RSV infection compared to the first RSV experienced subject.
 6. The method of claim 2, wherein the level of the RSV neutralizing antibodies in the first immunized subject reduces transmission of RSV infection from the first immunized subject.
 7. The method of claim 2, wherein the increase in the level of the RSV neutralizing antibodies in the first immunized subject is at least 100% compared to the level of RSV neutralizing antibodies in the first RSV experienced subject.
 8. The method of claim 1, wherein the first immunized subject comprises an increase in the level of the RSV neutralizing antibodies compared to the level of RSV neutralizing antibodies in a second RSV experienced subject that is infected with RSV.
 9. The method of claim 8, wherein the increase in the level of the RSV neutralizing antibodies in the first immunized subject is at least 100% compared to the level of RSV neutralizing antibodies in the second RSV experienced subject that is infected with RSV.
 10. The method of claim 1, wherein the first immunized subject comprises an increase in the level of the RSV neutralizing antibodies compared to the level of RSV neutralizing antibodies in a second immunized subject, wherein said second immunized subject is a second RSV experienced subject that is immunized with a second composition comprising chimeric ND VLPs that contain, in operable combination 1) stabilized post-fusion RSV F protein ectodomain, 2) TM domain of NDV F protein, and 3) CT domain of NDV F protein.
 11. (canceled)
 12. The method of claim 1, wherein the chimeric ND VLPs further comprise, in operable combination, foldon sequence listed as SEQ ID NO:14.
 13. The method of claim 1, wherein the level of the RSV neutralizing antibodies after a single administration of a dose of the first composition to the first RSV experienced subject is substantially the same as the level of RSV neutralizing antibodies after twice administering the dose of the first composition to a naïve subject.
 14. (canceled)
 15. The method of claim 1, further comprising comparing the level of the RSV neutralizing antibodies in the first immunized subject to the level of the RSV neutralizing antibodies in one or more test subjects selected from the group consisting of a) the first RSV experienced subject, b) a second RSV experienced subject that is infected with RSV, c) a second RSV experienced subject that is treated with a second composition comprising chimeric ND VLPs that contain, in operable combination 1) stabilized post-fusion RSV F protein ectodomain, 2) TM domain of NDV F protein, and 3) CT domain of NDV F protein, wherein detecting an increase in the level of the RSV neutralizing antibodies in the first immunized subject compared to the level of the RSV neutralizing antibodies in the one or more test subjects indicates that the first immunized subject is immunized against the RSV infection.
 16. The method of claim 1, further comprising detecting in the first immunized subject a reduction in one or more of (a) level of RSV infection, (b) one or more symptoms of RSV infection, (c) susceptibility to RSV infection, and (d) transmission of RSV infection, compared to the first RSV experienced subject.
 17. (canceled)
 18. The method of claim 1, wherein the ND VLP further comprises, in operable combination, CT domain of NDV FIN protein, TM domain of NDV HN protein, and RSV G ectodomain protein. 19-20. (canceled)
 21. The method of claim 1, wherein lung tissue of the first immunized subject contains a lower RSV titer than lung tissue of a naïve subject to which the first composition has not been administered.
 22. The method of claim 1, wherein the level of the RSV neutralizing antibodies after a single administration of the first composition to the first RSV experienced subject is higher than the level of RSV neutralizing antibodies after a single administration of the first composition to a naïve subject. 23-28. (canceled)
 29. A vaccine comprising recombinant chimeric Newcastle Disease virus-like particles (ND VLPs) that contain a chimeric protein comprising, in operable combination, 1) stabilized pre-fusion RSV F protein ectodomain, 2) transmembrane (TM) domain of NDV F protein, and 3) cytoplasmic (CT) domain of NDV F protein.
 30. The vaccine of claim 29, further comprising, in operable combination, foldon sequence listed as SEQ ID NO:14.
 31. The vaccine of claim 29, further comprising RSV G ectodomain protein sequence.
 32. The vaccine of claim 31, wherein the RSV G ectodomain protein sequence is operably linked to NDV HN TM domain and to NDV HN CT domain.
 33. The method of claim 1, wherein the level of said RSV neutralizing antibodies in said RSV experienced mammalian subject does not reduce RSV infection.
 34. The method of claim 1, wherein the level of said RSV neutralizing antibodies in said RSV experienced mammalian subject does not reduce symptoms of RSV infection.
 35. The method of claim 1, wherein the level of said RSV neutralizing antibodies in said RSV experienced mammalian subject does not reduce susceptibility to RSV infection.
 36. The method of claim 1, wherein the level of said RSV neutralizing antibodies in said RSV experienced mammalian subject does not reduce transmission of RSV infection from the first RSV experienced mammalian subject.
 37. The method of claim 1, wherein said administering comprises a single administration of said immunologically effective amount of said first composition. 38-40. (canceled)
 41. The method of claim 1, wherein said stabilized pre-fusion RSV F protein ectodomain comprising SEQ ID NO:06.
 42. The method of claim 10, wherein said stabilized post-fusion RSV F protein ectodomain comprises SEQ ID NO:08.
 43. The method of claim 15, wherein said stabilized post-fusion RSV F protein ectodomain comprises SEQ ID NO:08.
 44. The vaccine of claim 29, wherein said stabilized pre-fusion RSV F protein ectodomain comprises SEQ ID NO:06. 